Therapeutic variant alpha-2-macroglobulin compositions

ABSTRACT

A2M polypeptide compositions containing a non-natural bait region are disclosed. Methods of producing wild-type and variant A2M polypeptides and polynucleotides containing a non-natural bait region are also disclosed. The bait regions of the variant A2M polypeptides demonstrate enhanced protease inhibitory characteristics compared to wild-type A2M. Variant A2M polypeptides that demonstrate longer half-lives upon administration to an organism compared to wild-type A2M are disclosed. The A2M compositions are useful in treating a number of diseases and conditions including inflammation, chronic wounds, and diseases with a pathology associated with proteases.

CROSS-REFERENCE

This application claims priority to U.S. Provisional Application No.62/082,304, filed Nov. 20, 2014, which is incorporated herein byreference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 11, 2015, isnamed 37151-706.601_SL.txt and is 113,105 bytes in size.

BACKGROUND OF THE INVENTION

Inflammation causing spinal and joint pain can be difficult to treat.Increasing degrees of inflammation and force applied to joints result injoint injury. Abnormal joint anatomy can be a hallmark of aging, butjoint injury can be also a result of trauma, such as chondral lesionsoften seen in athletes. While joint injury resulting from trauma can betypically associated with acute inflammation, aberrant joint anatomyresulting from aging (e.g., osteoarthritis) can be a chronic condition.Physicians currently do not have a system or method available todifferentiate between acute injury due to trauma and age related jointdeteriorations.

Presently, it can be difficult to determine the appropriate course oftreatment for a given patient since it can be frequently unclear whetherthe particular condition the patient suffers from may be acute orchronic or if pathology in the joint is the cause of the pain.

Spinal-related pain can be typically classified as discogenic,facetogenic or radiculopathic pain. The manifestation of radiculopathicpain has traditionally been attributed to various physical and/ormechanical abnormalities, such as compression or mechanical irritationof the nerve root related to conditions such as disc herniation,stenosis, spondylolisthesis, sciatica, piriformis syndrome, obturatorsyndrome, cystic lesions (e.g., ganglion and synovial), tumors, andother pathology, such as chemically mediated causes.

Numerous studies have attempted to elucidate the pathophysiology ofspinal-related pain, and several molecular pathways have been implicatedtentatively. However, no clear causal pathway leading from injury ordegeneration to the painful state has been confirmed. Molecular markerscan be linked to clinical symptoms, and serve as potential targets forthe development of diagnostics and therapeutic tools. Although somestudies have provided evidence that the epidural space can be affectedby an intervertebral disc herniation, none has measured concentrationsof biomolecules in the epidural space in an attempt to detect thedifferences between affected and non-affected persons.

Tendons, which connect muscle to bone, and ligaments, which connectbones to other bones, are both composed of bands of fibrous connectivetissue. The cells of the fibrous connective tissue are mostly made up offibroblasts the irregular, branching cells that secrete strong fibrousproteins (such as collagens, reticular and elastic fibers, andglycoproteins) as an extracellular matrix. The extracellular matrix canbe defined in part as any material part of a tissue that is not part ofany cell. So defined, the extracellular matrix (ECM) can be thesignificant feature of the fibrous connective tissue.

The ECM's main component can be various glycoproteins. In most animals,the most abundant glycoprotein in the ECM can be collagen. Collagen canbe tough and flexible and gives strength to the connective tissue.Indeed, the main element of the fibrous connective tissue is collagen(or collagenous) fiber. The ECM also contains many other components:proteins such as fibrin and elastin, minerals such as hydroxyapatite, orfluids such as blood plasma or serum with secreted free flowingantigens. Given this diversity, it can serve any number of functions,such as providing support and anchorage for cells (which attach viafocal adhesions), providing a way of separating the tissues, andregulating intercellular communication. Therefore, the ECM can functionin a cell's dynamic behavior.

Injury to tendons and ligaments causes damage not only to the connectivetissue, but to the extracellular matrix as well. Damage to the ECM caninterrupt cell behavior in the connective tissue and decrease and/orlimit healing. After injury, continuing damage can be caused byproduction of matrix metalloproteinases (MMPs) by the body. MMPs areenzymes that degrade all components of the ECM. This can lead to animbalance between the synthesis and degradation of the ECM, as the bodytries to heal itself while the enzymes remodel the ECM. An overabundanceof remodeling by MMPs cause damage to previously connected tissue whichresults in the formation of scar tissue. In addition, scar tissueadhesion to surrounding tissue can cause further pulling and/orstretching of the tendons or ligaments and resultant pain.

Currently, treatment of injury to tendons and ligaments includes somesimple measures such as: avoiding activities that aggravate the problem;resting the injured area; icing the area the day of the injury; andtaking over-the-counter anti-inflammatory medicines. However, thesesimple remedies do not always cure the injury and often more advancedtreatments are needed. These treatments include: corticosteroidinjections, platelet-rich plasma (PRP), hyaluronic acid (HA) injection,physical therapy and even surgery. Corticosteroids are often usedbecause they can work quickly to decrease the inflammation and pain.Physical therapy can include range of motion exercises and splinting(such as for the fingers, hands, and forearm). Surgery can be onlyrarely needed for severe problems not responding to the othertreatments. It can be appreciated that additional treatment measures areneeded to treat and prevent extracellular matrix degradation for quickerand improved healing of tendons and ligaments.

Alpha-2-macroglobulin (A2M) is a highly conserved protease inhibitorpresent in plasma at relatively high concentrations (0.1-6 mg/ml). It isunique in its ability to inhibit all the major classes of proteases(Bhattacharjee et al (2000) J. Biol. Chem. 275, 26806-26811). A2M can beproduced by several cell types, such as hepatocytes, lung fibroblasts,macrophages, astrocytes and tumor cells (Borth W, “Alpha2-macroglobulin, A multifunctional binding and targeting protein withpossible roles in immunity and autoimmunity,” Ann. N.Y. Acad. Sci.737:267-272 (1994)). A2M often exists as a tetramer of four identical180 kDa subunits that forms a hollow cylinder-like structure. It canpresent multiple target peptide bonds to attacking proteases in itscentral “bait” domain. A2M can be the major protease inhibitor acting onforeign proteases, such as snake venoms. However, there are many otherprotease inhibitors in the circulation and it has been proposed that A2Mcan have other functions including binding to and regulation of cytokineand growth factor activity, promotion of tumoricidal capabilities ofmacrophages, and enhancement of antigen presentation. A2M can also be atargeting carrier for cytokines or growth factors.

SUMMARY OF THE INVENTION

Therefore, it is an object of the invention to provide compositions,systems, methods, and kits for the detection, diagnosis, and treatmentof inflammation, pain in the spine or joint, degradation ofextracellular matrix, and inhibiting fibronectin aggrecan complex (FAC)(FIG. 1). It is another object of the invention to provide biomarkersand methods for identifying sites in the spine or joint for treatingpain. It is another object of the invention to provide biomarkers thatcan be used to diagnose or assist in the diagnosis be of the presence ofpathologies that are causative of spinal- or joint-related pain. It isanother object of the invention to provide methods for diagnosing orassisting in the diagnosis of the presence of pathologies that arecausative of spinal- or joint related pain. Yet another object of theinvention is to provide biomarkers and methods to determine anappropriate therapy for a subject experiencing spinal- or joint-relatedpain. Another object of the invention is to provide biomarkers andmethods to monitor and assess the efficacy of a treatment for spinal- orjoint-related pain. Another object of the invention is to providecompositions and methods for treating spinal or joint pain and forselecting treatment sites in the spine or joint for treatment to inhibitor reduce pain.

Another object of the invention is to provide compositions, systems,methods, and kits for the detection, diagnosis, and treatment ofinflammation, degradation of extracellular matrix, and wounds. It isanother object of the invention to provide systems and methods toproduce compositions for the treatment of inflammation, degradation ofextracellular matrix, and chronic wounds. It is another object of theinvention to provide biomarkers and methods for identifying sites ofchronic wounds. It is another object of the invention to provide methodsfor diagnosing or assisting in the diagnosis of the presence ofpathologies that are causative of chronic wounds. Yet another object ofthe invention is to provide biomarkers and methods to determine anappropriate therapy for a subject experiencing chronic wounds. Anotherobject of the invention is to provide biomarkers and methods to monitorand assess the efficacy of a treatment for chronic wounds. Anotherobject of the invention is to provide compositions and methods fortreating chronic wounds and for selecting treatment sites and methodsfor treatment of chronic wounds.

Another object of the invention provides variant polypeptides fortreating chronic wounds. It is another object of the invention toprovide variant A2M polypeptides with a higher protease inhibitoryactivity than a wild-type A2M polypeptide. It is another object of theinvention to provide methods of making variant polypeptides for thetreatment of chronic wounds.

Another object of the invention provides variant polypeptides fortreating inflammation and pain. It is another object of the invention toprovide variant A2M polypeptides that inhibit the formation offibronectin aggrecan complex (FAC). Another object of the inventionprovides variant A2M polypeptides with a higher protease inhibitoryactivity than a wild-type A2M polypeptide. It is another object of theinvention to provide methods of making variant polypeptides for thetreatment of inflammation and pain.

In some aspects, compositions are provided that comprise a variant A2Mpolypeptide, comprising a bait region, wherein the bait region of thevariant A2M polypeptide comprises a plurality of protease recognitionsites arranged in series. In some embodiments, the variant A2Mpolypeptide is a recombinant protein. In some embodiments, the variantA2M polypeptide is produced in a host comprising bacteria, yeast, fungi,insect, or mammalian cells, or a cell free system. In some embodiments,the variant A2M polypeptide is characterized by an enhanced nonspecificinhibition of serine proteases, threonine proteases, cysteine proteases,aspartate proteases, metalloproteases, glutamic acid proteases, or anycombination thereof. In some embodiments, the variant A2M polypeptidefurther comprises PEG with abnormal glycosylation sites. In someembodiments, the variant A2M polypeptide has a longer half-life than thehalf-life of a wild type A2M protein when disposed within a joint orspine disc of a subject. In some embodiments, the plurality of proteaserecognition sites comprise one or more protease substrate bait regionsfrom one or more proteins other than A2M, one or more additionalprotease bait regions from A2M, one or more non-natural proteinsequences, or any combination thereof, wherein the modified A2M proteinis characterized by at least a 5%, 10%, 15%, 20%, 25%, or 30% increasein protease inhibitory effectiveness compared to the protease inhibitoryeffectiveness of a wild type A2M protein. In some embodiments, thenon-natural protein sequences comprise one or more protease recognitionsites that can function as bait for proteases. In some embodiments, theone or more protease substrate bait regions comprise consensus sequencesfor serine proteases, threonine proteases, cysteine proteases, aspartateproteases, metalloproteinases, glutamic acid proteases, or anycombination thereof. In some embodiments, the protease substrate baitregions comprise one or more consensus sequences for one or moreproteases from one or more organisms. In some embodiments, the one ormore organisms comprise animals, plants, bacteria, yeast, fish,reptiles, amphibians, or fungi. In some embodiments, one or more of theone or more protease substrate bait regions from the one or moreproteins other than A2M are the same. In some embodiments, one or moreof the one or more protease substrate bait regions from A2M are thesame. In some embodiments, one or more of the one or more proteasesubstrate bait regions from the one or more non-natural proteinsequences are the same. In some embodiments, one or more of the one ormore protease substrate bait regions from the one or more proteins otherthan A2M or from the one or more non-natural protein sequences comprisea suicide inhibitor; wherein the suicide inhibitor is operable tocovalently attach a protease to A2M. In some embodiments, one or more ofthe one or more protease substrate bait regions are from differentspecies.

In some aspects, provided herein is a composition comprising an isolatedvariant A2M polypeptide, wherein the variant A2M polypeptide comprisesone or more non-natural bait regions, wherein the one or morenon-natural bait regions comprise one or more protease recognition sitesnot present in a wild-type A2M polypeptide. In some embodiments, themodified A2M polypeptide is characterized by at least a 5%, 10%, 15%,20%, 25%, or 30% enhanced inhibition of one or more proteases comparedto a wild-type A2M inhibition of the one or more proteases. In someembodiments, the enhanced inhibition comprises enhanced nonspecificinhibition. In some embodiments, the enhanced inhibition comprisesenhanced specific inhibition. In some embodiments, the proteasecomprises a serine protease, threonine protease, cysteine protease,aspartate protease, metalloprotease, glutamic acid protease, or anycombination thereof. In some embodiments, the protease comprises MMP1(Interstitial collagenase), MMP2 (Gelatinase-A), MMP3 (Stromelysin 1),MMP7 (Matrilysin, PUMP 1), MMP8 (Neutrophil collagenase), MMP9(Gelatinase-B), MMP10 (Stromelysin 2), MMP11 (Stromelysin 3), MMP12(Macrophage metalloelastase), MMP13 (Collagenase 3), MMP14 (MT1-MMP),MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP18 (Collagenase 4,xcol4, Xenopus collagenase), MMP19 (RASI-1, stromelysin-4), MMP20(Enamelysin), MMP21 (X-MMP), MMP23A (CA-MMP), MMP23B, MMP24 (MT5-MMP),MMP25 (MT6-MMP), MMP26 (Matrilysin-2, endometase), MMP27 (MMP-22,C-MMP), MMP28 (Epilysin); A Disintegrin and Metalloproteinase withThrombospondin Motifs protease (ADAMTS), such as ADAMTS1, ADAMTS2,ADAMTS3, ADAMTS4, ADAMTS5 (ADAMTS11), ADAMTS6, ADAMTS7, ADAMTS8(METH-2), ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15,ADAMTS16, ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20; chymotrypsin; trypsin;elastase; compliment factors; clotting factors; thrombin; plasmin;subtilisin; Neprilysin; Procollagen peptidase; Thermolysin;Pregnancy-associated plasma protein A; Bone morphogenetic protein 1;Lysostaphin; Insulin degrading enzyme; ZMPSTE2; ZMPSTE4; ZMPSTE24;acetylcholinesterase; or a combination thereof. In some embodiments, theprotease comprises ADAMTS4, ADAMTS 5, MMP13, or a combination thereof.In some embodiments, the modified A2M polypeptide is characterized by atleast a 10% enhanced inhibition of FAC formation compared to a wild-typeA2M inhibition of FAC formation. In some embodiments, the one or morenon-natural bait regions are derived from one or more proteins otherthan A2M. In some embodiments, the one or more proteins other than A2Mare from a non-human organism. In some embodiments, the non-humanorganism comprises an animal, plant, bacterium, yeast, fish, reptile,amphibian, or fungi. In some embodiments, the one or more non-naturalbait regions comprise one or more sequences of SEQ ID NOs 6-83, orfragments thereof. In some embodiments, the variant A2M polypeptidecomprises SEQ ID NO 4, or a fragment thereof. In some embodiments, theone or more non-natural bait regions comprise SEQ ID NOs 6-30. In someembodiments, the one or more protease recognition sequences comprise SEQID NOs 31-83, or fragments thereof. In some embodiments, the wild-typeA2M polypeptide comprises SEQ ID NO 3, or a fragment thereof. Thewild-type A2M bait region consists of SEQ ID NO 5. In some embodiments,one or more of the one or more non-natural bait regions comprise asuicide inhibitor; wherein the suicide inhibitor is operable tocovalently attach a protease to the variant A2M polypeptide. In someembodiments, the one or more protease recognition sites comprise 2 ormore copies of the one or more protease recognition sequences. In someembodiments, the one or more non-natural bait regions comprise 2 or morecopies of the one or more non-natural bait regions. In some embodiments,the variant A2M polypeptide comprises a wild-type A2M bait regionsequence. In some embodiments, the variant A2M polypeptide is arecombinant polypeptide. In some embodiments, the one or more proteaserecognition sites comprise a consensus sequence for a protease. In someembodiments, the variant A2M polypeptide comprises one or more modifiedglycosylation sites. In some embodiments, the one or more modifiedglycosylation sites are functionalized with PEG. In some embodiments,the variant A2M polypeptide has at least a 10% longer half-life than thehalf-life of a wild type A2M polypeptide when disposed within a subject.

In some aspects, provided herein is a method of treating a subject withone or more conditions, comprising administering to the subject aneffective amount of any composition provided herein comprising an A2Mvariant. In some embodiments, nonspecific inhibition of one or moreproteases in the subject, inhibition Aggrecan G3 fragment formation,inhibition FAC formation, or a combination thereof, is increased. Insome embodiments, the rate of degeneration of tissue, cartilage anddiscs, synovial inflammation, or a combination thereof, is decreased inthe subject. In some embodiments, treating results in a reduction inseverity, occurrence, rate of progression, or a combination thereof, ofthe one or more conditions. In some embodiments, any of the methodsprovided herein further comprise administering one or more additionalcarriers or drugs. In some embodiments, the one or more additionalcarriers or drugs comprise hydrogels, hyaluronic acid preparations,polymer microspheres, corticosteroids, microparticles, chitosan, localanesthetics, growth factors, cytokines, protease inhibitors, steroids,hyaluronic Acid (HA), or other biologically active autogenous orendogenous mediators. In some embodiments, the one or more conditionsare treatable with any composition provided herein. In some embodiments,the one or more conditions comprise cancer, degenerative diseases,traumatic diseases, and/or inflammatory diseases, whose pathogenesisincludes the activity of proteases. In some embodiments, the cancer,degenerative diseases, traumatic diseases, and/or inflammatory diseaseswhose pathogenesis includes the activity of proteases comprisesosteoarthritis, inflammatory arthritides, chondrosis, chondral injuries,enthesopathies, tendinopathies, ligamentous injuries, degenerativediseases of the bone, cartilage, tendons, and ligaments, post-operativeconditions and wound healing, and other musculoskeletal diseases. Insome embodiments, the one or more conditions comprise cancer, arthritis,inflammation, ligament injury, tendon injury, bone injury, cartilagedegeneration, cartilage injury, an autoimmune disease, back pain, jointpain, joint degeneration, disc degeneration, spine degeneration, bonedegeneration, or any combination thereof. In some embodiments,inflammation comprises joint or disc inflammation caused by surgery,joint or disc inflammation caused by a joint or disc replacement, or acombination thereof. In some embodiments, the subject is a human, pig,mouse, rat, rabbit, cat, dog, monkey, frog, horse or goat. In someembodiments, the subject has been previously diagnosed with the one ormore conditions. In some embodiments, the composition is administeredinto an anatomic site relevant to the host pathology. In someembodiments, the administration comprises injection with a hollow-lumendevice or flexible catheter combinations. In some embodiments, thehollow-lumen device comprises a needle, syringe, or combination thereof.In some embodiments, the administration occurs during a surgicalprocedure.

In some aspects, provided herein is a composition comprising an isolatedvariant A2M polynucleotide, wherein the variant A2M polynucleotideencodes for one or more non-natural bait regions, wherein the one ormore non-natural bait regions comprise one or more protease recognitionsites not present in a wild-type A2M polypeptide. In some embodiments,the non-natural bait regions comprise a sequence with at least 60%, 70%,80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% identity to any one of SEQID NOs 6-83, or fragments thereof. In some embodiments, the non-naturalbait regions comprise one or more protease recognition sequences with atleast 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% identityto any one of SEQ ID NOs 31-83. In some embodiments, the variant A2Mpolynucleotide comprises at least 90% identity to SEQ ID NO 2, or afragment thereof. In some embodiments, the wild-type A2M polynucleotidecomprises SEQ ID NO 1, or a fragment thereof. In some embodiments, thevariant A2M polynucleotide is within an expression vector.

In one aspect, provided herein is a method for determining the enhancedinhibition of a protease by a variant A2M polypeptide comprising: (a)providing a variant A2M polypeptide comprising a sequence of one or moreof SEQ ID NOs 6-83; (b) contacting the variant A2M polypeptide with theprotease and a substrate cleaved by the protease; (c) contacting awild-type A2M polypeptide with the protease and the substrate cleaved bythe protease; and (d) comparing the amount of cleavage of the substratefrom step (b) to the amount of cleavage of the substrate from step (c),thereby determining the enhanced inhibition of the protease by thevariant A2M polypeptide.

In some aspects, provided herein is a method for making a variant A2Mpolynucleotide comprising: (a) providing a vector containing a variantA2M polynucleotide comprising a sequence of SEQ ID NO 2; (b) digestingthe vector containing a variant A2M polynucleotide with restrictionendonucleases to form a linear vector; (c) ligating one end of the oneor more polynucleotides encoding one or more of the non-natural baitregions of SEQ ID NOs 6-83 to one end of the linear vector; and (d)ligating the other end of the one or more polynucleotides encoding oneor more of the non-natural bait regions of SEQ ID NOs 6-83 to the otherend of the linear vector, thereby forming a vector containing a variantA2M polynucleotide comprising the non-natural bait regions of SEQ ID NOs6-83.

In some aspects, provided herein is a method for making a variant A2Mpolynucleotide comprising: (a) providing a vector containing a variantA2M polynucleotide comprising a sequence of SEQ ID NO 2; (b) digestingthe vector containing a variant A2M polynucleotide with restrictionendonucleases to form a linear vector; (c) ligating one end of the oneor more polynucleotides encoding one or more of the non-natural baitregions of SEQ ID NOs 6-30 or one or more protease recognition sites ofSEQ ID NOs 31-83 to one end of the linear vector; and (d) ligating theother end of the one or more polynucleotides encoding one or more of thenon-natural bait regions of SEQ ID NOs 6-30 or one or more proteaserecognition sites of SEQ ID NOs 31-83 to the other end of the linearvector, thereby forming a vector containing a variant A2M polynucleotidecomprising the non-natural bait regions of SEQ ID NOs 6-30 or one ormore protease recognition sites of SEQ ID NOs 31-83.

In some aspects, a composition is provided that comprises a variant A2Mpolypeptide or polynucleotide, wherein the composition is obtainable byany method provided herein.

In one aspect, provided herein is a composition comprising A2M for usein therapy wherein the composition is a composition obtainable by anymethod provided herein, or any variant A2M composition provided hereinIn some embodiments, the composition is for use in the treatment ofcancer, arthritis, inflammation, ligament injury, tendon injury, boneinjury, cartilage degeneration, cartilage injury, an autoimmune disease,back pain, joint pain, joint degeneration, disc degeneration, spinedegeneration, bone degeneration, or any combination thereof; whereininflammation comprises joint or disc inflammation caused by surgery,joint or disc inflammation caused by a joint or disc replacement, or acombination thereof.

In one aspect, provided herein is a use of a composition obtainable byany method provided herein, or any variant A2M composition providedherein, for the manufacture of a medicament for use in therapy. In someembodiments, the medicament is for use in the treatment of cancer,arthritis, inflammation, ligament injury, tendon injury, bone injury,cartilage degeneration, cartilage injury, an autoimmune disease, backpain, joint pain, joint degeneration, disc degeneration, spinedegeneration, bone degeneration, or any combination thereof; whereininflammation comprises joint or disc inflammation caused by surgery,joint or disc inflammation caused by a joint or disc replacement, or acombination thereof.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.In the event of a conflict between a term herein and a term incorporatedby reference, the term herein controls.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features are set forth with particularity in the appendedclaims. A better understanding of the features and advantages will beobtained by reference to the following detailed description that setsforth illustrative embodiments, in which the principles of devices,methods, and compositions are utilized, and the accompanying drawings ofwhich:

FIG. 1 depicts a schematic of the steps and signaling pathwaysassociated with formation of a fibronectin-aggrecan complex (FAC) andthe FAC-induced activation of Damage-Associated-Molecular Pattern (DAMP)receptor signaling in cells. The combination of the two processescreates a cyclic process that continually degrades cartilage.

FIG. 2 depicts FAC formation using fibronectin to form a complex withpurified full length Aggrecan or recombinant G3 Aggrecan. Both Aggrecanand the G3 domain bind fibronectin to form FAC.

FIG. 3 depicts a flow chart of the steps for construct or proteinexpression.

FIG. 4 depicts the A2M structure and various domains of A2M.

FIG. 5A depicts a graph demonstrating treatment of Bovine CartilageExplants (BCE) with leukocyte-rich Platelet Rich Plasma (LR-PRP), whichinduces cartilage catabolism, and treatment with purified A2M to inhibitcartilage degradation.

FIG. 5B depicts a graph demonstrating treatment of Bovine CartilageExplants (BCE) with APIC-PRP, blood, or leukocyte-rich Platelet RichPlasma (LR-PRP) from the same patient. LR-PRP, but not blood, inducescartilage catabolism. Treatment of BCE with APIC-PRP inhibits cartilagedegradation below endogenous levels.

FIG. 5C depicts a graph demonstrating leukocyte-rich Platelet RichPlasma (LR-PRP) induces cartilage catabolism in a Bovine CartilageExplant (BCE) model. Treatment with APIC-PRP inhibits the cartilagedegradation induced by treatment with LR-PRP.

FIG. 6A depicts a graph showing Bovine Cartilage Explants (BCE) treatedwith pro-inflammatory cytokines TNF-α and IL-1β to induce cartilagecatabolism. Cartilage catabolism with each cytokines separately isdemonstrated by the release of sulfated Glycosaminoglycans (sGAG) intothe culture media. Treatment with APIC-PRP efficiently inhibitscartilage catabolism by each pro-inflammatory cytokine separately.

FIG. 6B depicts a graph showing Bovine Cartilage Explants (BCE) treatedwith the combination of pro-inflammatory cytokines TNF-α and IL-β toinduce cartilage catabolism. Treatment with APIC-PRP efficientlyinhibited cartilage catabolism by the combination of pro-inflammatorycytokines in a dose dependent manner.

FIG. 7A depicts the sulfated glycosaminoglycan (sGAG) released uponcartilage catabolism in a BCE model with and without treatment ofADAMTS-5 and treatment with or without a serial dilution of purified A2M(top). Western Blots of the samples (bottom) demonstrate ADAMTS-5degradation of cartilage produced an Aggrecan G3 fragment and highermolecular weight Aggrecan fragments, which were inhibited by treatmentwith A2M in a dose dependent manner. Values above the columns indicatethe concentration of A2M (μg/ml) needed to inhibit ADATMS-5. An 85 kDanon-specific band is also visible, which was apparent in media-onlycontrols (data not shown).

FIG. 7B depicts the sulfated glycosaminoglycan (sGAG) released uponcartilage catabolism in a BCE model with and without treatment ofADAMTS-4 and treatment with or without a serial dilution of purified A2M(top). Western Blot analysis with α-Aggrecan G3 antibody (bottom) of thesamples demonstrates ADAMTS-4 degradation of cartilage produced highmolecular weight Aggrecan C-terminal fragments containing the G3 domain.Cartilage catabolism is inhibited by A2M in a dose dependent manner andreduces the release of cartilage aggrecan fragments. An 85 kDanon-specific band is also visible, which was apparent in media-onlycontrols (data not shown).

FIG. 8A depicts a graph demonstrating the sulfated glycosaminoglycan(sGAG) released upon cartilage catabolism in a BCE model with andwithout treatment of MMP-7 and MMP-12. Treatment with purified A2Minhibited the MMP-induced cartilage catabolism.

FIG. 8B depicts a stained SDS-PAGE gel of samples produced in FIG. 9A.The MMP-7- or MMP-12-induced degradation of cartilage, and theproduction of cartilage protein fragments visible in the gel, wasinhibited with addition of purified A2M.

FIG. 8C depicts a Western Blot with α-Aggrecan G3 antibody using the gelfrom FIG. 8B and the samples from FIG. 8A. The degradation of cartilageby MMP-7 or MMP-12 produces an Aggrecan G3 fragment at ˜30 kDa which canbe inhibited with addition of purified A2M.

FIG. 9 depicts the results of an ELISA test that recognizes complexes ofFibronectin and Aggrecan G3 (FACT, Fibronectin Aggrecan Complex Test).Culture media from BCE treated with or without the listed proteases inthe presence or absence of A2M were incubated with Synovial Fluid (SF)spiked with free Fibronectin and tested on the FACT assay. In each casewhere degradation of cartilage led to Aggrecan fragments the result wasformation of additional Fibronectin Aggrecan Complexes above the SFbackground control. Treatment with A2M, however, which preventedcartilage catabolism, subsequently preventing FAC formation.

FIG. 10 depicts two bar graphs demonstrating the ability of APIC(Retentate from the 500 kDa filter) and the Filtrate to preventcartilage degradation. Cartilage catabolism was induced in the BCE modelwith ADAMTS-5, which could be inhibited with serial dilution of APIC(left, Retentate), but not the Filtrate which is devoid of A2M (right,Filtrate). The numbers above the columns represent the percentage ofAPIC (v/v) or filtrate in the culture media. The inhibitory potential in5% of Filtrate is equivalent to 0.01% of APIC; thus the process ofproducing APIC concentrates >99% of the chondroprotective effects ofblood.

FIG. 11 is a bar graph depicting the effects of treatment of THP-1monocytes with variant A2M for two days in culture. No activation of themonocytes was observed through monitoring with a panel of cytokines,chemokines, and growth factors (Left to right: IL-β, IL-1 receptoragonist (IL-1ra), IL-6, IFN-γ, IP-10, MCP-10, MIP-β, PDGF-ββ, RANTES,TNF-α, and VEGF).

FIG. 12 depicts macroscopic images of rabbit knees 6 weeks after ACL-Tsurgery and treatment with saline or APIC cell free. Sham surgerieswithout ACL-T were performed as a control.

FIG. 13A depicts a graph of macroscopic evaluation for the experimentsshown in FIG. 12. The values shown are the average of the macroscopicevaluation of 6 rabbits.

FIG. 13B depicts a graph of macroscopic evaluation, showing an inversecorrelation of A2M in APIC cell free treatment and cartilage degradationfor the experiments shown in FIG. 12.

FIG. 14 depicts graphs of histopathology evaluation of the rabbit kneesfrom experiments depicted in FIGS. 12 and 13 including structure,chondrocyte density, Safarin-O staining, and cluster formationevaluations; and shows an inverse correlation between A2M concentrationin each rabbit's APIC and the scoring criteria. One outlying rabbit isexcluded from calculations in the line but is included in the figures.

FIG. 15 is a depiction of a pseudocolored stain-free SDS-PAGE gel of arepresentative purification of tagged wild-type A2M and the fourselected variable bait region A2M proteins. The theoretical molecularweight of a monomer of wild-type A2M is 163 KDa, not includingglycosylation. The blurry band above 250 KDa is comprised of dimeric A2Mthat is not thoroughly reduced during sample preparation or covalentlybound dimer through amino acid modification mechanisms.

FIG. 16 is a depiction of a pseudocolored stain-free SDS-PAGE gel (top)and Western blot (bottom) of a representative screening assay forinhibition of ADAMTS-5 cleavage of aggrecan IGD domain (IGD fragment) bywild-type (WT) and bait region substituted A2M. The negative control isIGD fragment protein alone; the positive control is IGD fragment plusADAMTS-5. ADAMTS-5, Wild-type and variant A2M were each kept at 50 nM,and the A2M and ADAMTS-5 were pre-mixed for 10 min. before addition ofIGD fragment. The primary antibody for the Western blot was ananti-Aggrecan G1-IGD-G2 polyclonal antibody (R&D).

FIG. 17 is a graph depicting a comparison of the relative inhibitorycharacteristics of the four chosen variants vs. various MMPs andADAMTS-4 and -5 as determined by the two IGD screening experiments. Ineach case the unit for the y-axis is multiples of the wild-typeinhibition of each protease.

FIG. 18A depicts the raw data (left) and calculated slope (right) ofdigestion of FTC-casein by bovine trypsin in the presence of taggedwild-type A2M (WT) or the four chosen A2M variants. The samples withoutthe “-D” are prepared with a 1:1 molar ratio of A2M:protease. Those withthe “-D” are prepared at a 0.5:1 ratio of A2M:protease.

FIG. 18B depicts the raw data (left) and calculated slope (right) ofdigestion of FTC-casein by chymotrypsin in the presence of taggedwild-type A2M (WT) or the four chosen A2M variants. The samples withoutthe “-D” are prepared with a 1:1 molar ratio of A2M:protease. Those withthe “-D” are prepared at a 0.5:1 ratio of A2M:protease.

FIG. 19 depicts a western blot analysis of a cleavage assay using IGDfragment as a substrate in the presence of the MMP3.

FIG. 20 depicts a chart of the inhibition of IGD fragment proteolysis bythe indicated variants as a percentage of wild-type A2M (top) and thesequences of the bait sequences corresponding to the indicated A2Mvariants (bottom) (SEQ ID NOS 152-155, respectively, in order ofappearance).

FIG. 21 depicts Western blots showing the control blot of degraded andnon-degraded forms of A2M as a function of the known amount of proteinindicated (top) and the cleavage of various A2M polypeptides over timein the presence of a protease (bottom). The control blot can be used toquantify the amount of cleaved A2M, which is directly proportional tothe rate of protease inhibition.

FIG. 22 depicts the protective effect of the A2M wild type vs. some ofthe variants of the digestion of IGD domain from a mixture of proteases.10 nM of each MMP1, MMP3, MMP7, MMP13, ADAMTS4 and ADAMTS5 were mixedand used to digest IGD in the presence or absence of A2M wild type andA2M variants.

FIG. 23 depicts a Vector Map of pJ608 mammalian expression vector. TheORF sequence coding for wild-type and variant A2M is cloned in betweenthe Kpn1 and BamH1 restriction sites.

DETAILED DESCRIPTION OF THE DISCLOSURE

Provided herein are compositions, methods, kits and systems for thedetection, diagnosis, and treatment of inflammation, pain in the spineor joint, and degradation of extracellular matrix.

The details of one or more inventive embodiments are set forth in theaccompanying drawings, the claims, and in the description herein. Otherfeatures, objects, and advantages of inventive embodiments disclosed andcontemplated herein will be apparent from the description and drawings,and from the claims. As used herein, unless otherwise indicated, thearticle “a” means one or more unless explicitly otherwise provided for.As used herein, unless otherwise indicated, terms such as “contain,”“containing,” “include,” “including,” and the like mean “comprising.” Asused herein, unless otherwise indicated, the term “or” can beconjunctive or disjunctive. As used herein, unless otherwise indicated,any embodiment can be combined with any other embodiment. As usedherein, unless otherwise indicated, some inventive embodiments hereincontemplate numerical ranges. When ranges are present, the rangesinclude the range endpoints. Additionally, every subrange and valuewithin the range is present as if explicitly written out.

Definitions

The term “non-immunogenic” or “non-antigenic” means that the compositionbeing administered to a subject does not elicit an immune response.

A “subject” refers to a donor, recipient or host of the composition ofthe present invention. In some embodiments, the donor and the recipientare the same. In some embodiments the subject is a human subject.

A “proteoglycan” refers to a special class of proteins that are heavilyglycosylated. A proteoglycan is made up of a core protein with numerouscovalently attached high sulphated glycosaminoglycan chain(s).Non-limiting example of extracellular matrix proteoglycans includeaggrecan and certain collagens, such as collagen IX.

A “glycosaminoglycan” or “GAG” as used herein refers to a longunbranched polysaccharide molecules found on the cell surface or withinthe extracellular matrix. Non-limiting examples of glycosaminoglycaninclude heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate,heparin sulfate, keratin sulfate, hyaluronic acid, hexuronylhexosaminoglycan sulfate, and inositol hexasulfate.

The term “non-autologous” refers to tissue or cells which originate froma donor other than the recipient. Non-autologous can refer to, forexample, allogeneic or xenogeneic. The term “autologous” as in anautologous composition, refers to a composition in which the donor andrecipient is the same individual. Likewise, “allogeneic” refers to adonor and a recipient of the same species; “syngeneic” refers to a donorand recipient with identical genetic make-up (e.g. identical twins orautogeneic) and “xenogeneic” refers to donor and recipient of differentspecies.

“Variant” (or “analog”) refers to a molecule differing from thewild-type molecule.

The term “variant polynucleotide” (or “analog”) refers to anypolynucleotide differing from the naturally occurring polynucleotide.For example, “variant A2M polynucleotide” refers to any A2Mpolynucleotide differing from naturally occurring A2M polynucleotides. Avariant A2M polynucleotide includes a polynucleotide sequence differentfrom the wild-type A2M polynucleotide sequence (SEQ ID NO: 1). Variantpolynucleotides can be characterized by nucleic acid insertions,deletions, and substitutions, created using, for example, recombinantDNA techniques. A variant A2M polynucleotide preferably includes amutation, insertion, deletion, or a combination thereof, in the baitregion of a wild-type A2M polynucleotide sequence. As used herein, whenreferring to polypeptides, the “bait region” includes the region of anA2M polynucleotide that encodes the region of the A2M polypeptide thatbinds to proteases, for example, regions that contain proteaserecognition sites. A variant A2M polynucleotide includes an “A2Macceptor sequence” (SEQ ID NO: 2) which includes a polynucleotidesequence of A2M with point mutations that can aid in creating variantA2M polynucleotides by recombinant DNA techniques, for example, bycreating restriction enzyme cloning sites to aid in inserting variouspolynucleotide sequences encoding the variant bait regions. Variant baitregions can include one or more sequences of SEQ ID NOs: 6-83 andsequences substantially similar to SEQ ID NOs: 6-83. For example,variant bait regions can include one or more nonnatural bait regions ofSEQ ID NOs 6-30, one or more protease recognition sites of SEQ ID NOs31-83, or any combination thereof.

The term “variant polypeptide” refers to any polypeptide differing fromthe naturally occurring polypeptide. For example, “variant A2Mpolypeptide” refers to any A2M polypeptide differing from naturallyoccurring A2M polypeptides. Variant polypeptides can be characterized byamino acid insertions, deletions, and substitutions, created using, forexample, recombinant DNA techniques. A variant A2M polypeptide includesa polypeptide sequence different from the wild-type A2M polypeptidesequence. A variant A2M polypeptide preferably includes a mutation,insertion, deletion, or a combination thereof, in the bait region of awild-type A2M protein. When referring to polypeptides, the “bait region”includes the region of an A2M polypeptide that binds to proteases, forexample, a stretch of amino acids that contains one or more proteaserecognition sites. A variant A2M polypeptide includes a polypeptide (SEQID NO: 3) encoded by an A2M acceptor sequence (SEQ ID NO: 2). A “variantA2M polypeptide” can have at least one amino acid sequence alteration inthe bait region as compared to the amino acid sequence of thecorresponding wild-type polypeptide. An amino acid sequence alterationcan be a substitution, a deletion, or an insertion of one or more aminoacids. A variant A2M polypeptide can have any combination of amino acidsubstitutions, deletions or insertions.

Guidance in determining which amino acid residues may be replaced, addedor deleted without abolishing activities of interest, may be found bycomparing the sequence of the particular polypeptide with that ofhomologous peptides and minimizing the number of amino acid sequencechanges made in regions of high homology (conserved regions) or byreplacing amino acids with consensus sequence. Alternatively,recombinant variants encoding these same or similar polypeptides may besynthesized or selected by making use of the “redundancy” in the geneticcode. Various codon substitutions, such as the silent changes whichproduce various restriction sites, may be introduced to optimize cloninginto a plasmid or viral vector or expression in a particular prokaryoticor eukaryotic system. Mutations in the polynucleotide sequence may bereflected in the polypeptide or domains of other peptides added to thepolypeptide to modify the properties of any part of the polypeptide, tochange characteristics such as inhibition of proteases, ligand-bindingaffinities, interchain affinities, or degradation/turnover rate. Variantnucleotides can also be used to generate polypeptides that are bettersuited for expression, scale up and the like in the host cells chosenfor expression. For example, cysteine residues can be deleted orsubstituted with another amino acid residue in order to eliminatedisulfide bridges.

An amino acid “substitution” includes replacing one amino acid withanother amino acid having similar structural and/or chemical properties,for example, conservative amino acid replacements. “Conservative” aminoacid substitutions can be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, the amphipathicnature of the residues involved, or a combination thereof. Nonpolar(hydrophobic) amino acids include alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. Polar neutral aminoacids include glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine. Positively charged (basic) amino acidsinclude arginine, lysine, and histidine. Negatively charged (acidic)amino acids include aspartic acid and glutamic acid. “Insertions” or“deletions” are preferably in the range of about 1 to 50 amino acids,more preferably 1 to 30 amino acids. The variation allowed can beexperimentally determined by inserting, deleting, or substituting aminoacids in a polypeptide using recombinant DNA techniques and assaying theresulting recombinant variants for activity, for example, proteaseinhibition activity.

The terms “purified” or “substantially purified” as used herein denotesthat the indicated nucleic acid or polypeptide is present in thesubstantial absence of other biological macromolecules, for example,polynucleotides, proteins, and the like. The polynucleotide orpolypeptide can be purified such that it constitutes at least 95% byweight, for example, at least 99% by weight, of the indicated biologicalmacromolecules present. Water, buffers, and other small molecules with amolecular weight of less than 1000 Daltons, can be present in anyamount. The term “isolated” as used herein refers to a polynucleotide orpolypeptide separated from at least one other component present with thepolynucleotide or polypeptide in its natural source. In someembodiments, the polynucleotide or polypeptide can be found in thepresence of only a solvent, buffer, ion, or other components normallypresent in a solution of the same. The terms “isolated” and “purified”do not encompass polynucleotides or polypeptides present in theirnatural source.

As used herein, “recombinant polypeptides” include polypeptides orproteins derived from recombinant expression systems, for example,microbial, insect, or mammalian expression systems. Polypeptides orproteins expressed in most bacterial cultures will be free ofglycosylation modifications; polypeptides or proteins expressed in yeastcan have a glycosylation pattern in general different from thoseexpressed in mammalian cells.

The term “expression vector” refers to a plasmid or phage or virus orvector, for expressing a polypeptide from a DNA or RNA sequence. Anexpression vector can include a transcriptional unit comprising anassembly of a genetic element or elements having a regulatory role ingene expression, for example, promoters or enhancers, a structural orcoding sequence which is transcribed into mRNA and translated intoprotein, and appropriate transcription initiation and terminationsequences. Structural units intended for use in yeast or eukaryoticexpression systems can include a leader sequence enabling extracellularsecretion of translated protein by a host cell. Alternatively, whererecombinant protein is expressed without a leader or transport sequence,it can include an amino terminal methionine residue. This residue may besubsequently cleaved from the expressed recombinant protein to provide afinal product.

The term “recombinant expression system” means host cells which havestably integrated a recombinant transcriptional unit into chromosomalDNA or carry the recombinant transcriptional unit extrachromosomally.Recombinant expression systems can be used to express heterologouspolypeptides or proteins upon induction of the regulatory elementslinked to the DNA segment or synthetic gene to be expressed. This termincludes host cells which have stably integrated a recombinant geneticelement or elements having a regulatory role in gene expression, forexample, promoters or enhancers. Recombinant expression systems can beused to express polypeptides or proteins endogenous to the cell uponinduction of the regulatory elements linked to the endogenous DNAsegment or gene to be expressed. The cells can be prokaryotic oreukaryotic.

The term “secreted” includes a protein that is transported across orthrough a membrane, including transport as a result of signal sequencesin its amino acid sequence when it is expressed in a suitable host cell.“Secreted” proteins include without limitation proteins secreted wholly,for example soluble proteins, or partially, for example receptors, fromthe cell in which they are expressed. “Secreted” proteins also includeproteins transported across the membrane of the endoplasmic reticulum.“Secreted” proteins also include those non-typical signal sequences.

Where desired, an expression vector may be designed to contain a “signalsequence” which will direct the polypeptide through the membrane of acell. A signal sequence can be naturally present on the polypeptides orprovided from heterologous protein sources.

As used herein, “substantially equivalent” or “substantially similar”can refer both to nucleotide and amino acid sequences, for example avariant sequence, that varies from a reference sequence by one or moresubstitutions, deletions, or additions, the net effect of which does notresult in an adverse functional dissimilarity between the reference andsubject sequences. Typically, such a substantially equivalent sequencevaries from one of those listed herein by no more than about 35%. Forexample, the number of individual residue substitutions, additions,and/or deletions in a substantially equivalent sequence, as compared tothe corresponding reference sequence, divided by the total number ofresidues in the substantially equivalent sequence is about 0.35 or less.A substantially equivalent sequence includes sequences with 65% sequenceidentity to the reference sequence. A substantially equivalent sequenceof the invention can vary from a reference sequence by no more than 30%(70% sequence identity), no more than 25% (75% sequence identity), nomore than 20% (80% sequence identity), no more than 10% (90% sequenceidentity), or no more that 5% (95% sequence identity). Substantiallyequivalent amino acid sequences according to the invention preferablyhave at least 80% sequence identity with a reference amino acidsequence, at least 85% sequence identity, at least 90% sequenceidentity, at least 95% sequence identity, at least 98% sequenceidentity, or at least 99% sequence identity. Substantially equivalentpolynucleotide sequences of the invention can have lower percentsequence identities, taking into account, for example, the redundancy ordegeneracy of the genetic code. Preferably, the polynucleotide sequencehas at least about 65%, at least about 75%, at least about 80%, at least85%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. Sequences having substantially equivalent biological activityand substantially equivalent expression characteristics are consideredsubstantially equivalent. Identity between sequences can be determinedby methods known in the art, such as by alignment of the sequences orvarying hybridization conditions.

As used herein the term “effective amount” or “therapeutically effectiveamount” means a dosage sufficient to treat, inhibit, or alleviate spinalpain in a subject in need thereof.

By “degenerate variant” can be intended nucleotide fragments whichdiffer from a nucleic acid fragment of the present invention (e.g., anORF) by nucleotide sequence but, due to the degeneracy of the geneticcode, encode an identical polypeptide sequence.

The terms “polypeptide”, “peptide”, and “protein” can be usedinterchangeably and can refer to a polymer of amino acid residues or avariant thereof. Amino acid polymers can have one or more amino acidresidues and can be an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers, those containing modified residues, and non-naturallyoccurring amino acid polymers. A variant polypeptide can have at leastone amino acid sequence alteration as compared to the amino acidsequence of the corresponding wild-type polypeptide. An amino acidsequence alteration can be, for example, a substitution, a deletion, oran insertion of one or more amino acids. A variant polypeptide can haveany combination of amino acid substitutions, deletions or insertions. Anamino acid sequence alteration can be formed by altering the nucleotidesequence from which it is derived, such as a mutation, for example, aframeshift mutation, nonsense mutation, missense mutation, neutralmutation, or silent mutation. For example, sequence differences, whencompared to a wild-type nucleotide sequence, can include the insertionor deletion of a single nucleotide, or of more than one nucleotide,resulting in a frame shift; the change of at least one nucleotide,resulting in a change in the encoded amino acid; the change of at leastone nucleotide, resulting in the generation of a premature stop codon;the deletion of several nucleotides, resulting in a deletion of one ormore amino acids encoded by the nucleotides; the insertion of one orseveral nucleotides, such as by unequal recombination or geneconversion, resulting in an interruption of the coding sequence of areading frame; duplication of all or a part of a sequence;transposition; or a rearrangement of a nucleotide sequence. Suchsequence changes can alter the polypeptide encoded by the nucleic acid,for example, if the change in the nucleic acid sequence causes a frameshift, the frame shift can result in a change in the encoded aminoacids, and/or can result in the generation of a premature stop codon,causing generation of a truncated polypeptide.

The term “fragment” can refer to any subset of the polypeptide that canbe a shorter polypeptide of the full length protein. Fragments of A2Mcan include 20, 30, 40, 50 or more amino acids from A2M that can bedetected with anti-A2M antibodies. Other fragments of A2M includevarious domains of A2M and combinations thereof.

“Platelet-rich plasma” (“PRP”) refers to blood plasma enriched withplatelets.

Variant A2M Polypeptides Compositions for Therapy

A2M is a general inhibitor of metalloproteases and other proteases suchas ADAMTS 4 and ADAMTS 5. These proteases and others produced as aresult of or prior of degeneration and inflammation can be responsiblefor cartilage and disc degeneration and pain in synovial joints, thespine, tendons and ligaments, and other joints, entheses and generaltissues. Any of the recombinant compositions described herein can beused for treatment of a subject with a condition, disease, pain orinflammation according to any of the methods described herein.

A2M is able to inactivate an enormous variety of proteases (includingserine-, cysteine-, and aspartic-metalloproteases). A2M can function asan inhibitor of fibrinolysis by inhibiting plasmin and kallikrein. A2Mcan function as an inhibitor of coagulation by inhibiting thrombin.Human A2M has in its structure a 38 amino acid “bait” region. The baitregion varies widely in the amino acid number (27-52 amino acids) andsequence between animal species. Proteases binding and cleaving of thebait region can become bound to A2M. The protease-A2M complex can berecognized by macrophage receptors and cleared from the organism'ssystem. A2M is able to inhibit all four classes of proteases by a unique‘trapping’ mechanism. When a protease cleaves the bait region, aconformational change can be induced in the protein which can trap theprotease. The entrapped enzyme can remain active against low molecularweight substrates (activity against high molecular weight substrates canbe greatly reduced). Following cleavage in the bait region a thioesterbond can be hydrolyzed and can mediate the covalent binding of theprotein to the protease.

In one aspect, provided herein is a composition that can be a variantA2M polypeptide. A variant A2M polypeptide can be a recombinant protein,or fragments thereof, and can be produced in a host cell and purifiedfor use in treatment of pain and inflammation conditions and diseases. Avariant A2M composition can be more efficient in inhibiting proteases,have longer half-life, have a slower clearance factor, or anycombination thereof compared to a wild-type A2M. A variant A2M can be arecombinant protein, or a fragment thereof, and can be produced in ahost cell and purified. For example, a variant A2M recombinant proteincan be produced in a host comprising bacteria, yeast, fungi, insect, ormammalian cells, or a cell free system.

Variant A2M polypeptides or fragments thereof, can also be variants orposttranslationally modified variants of A2M. A2M variant polypeptidescan have an integer number of amino acid alterations such that theiramino acid sequence shares at least about 60%, 70%, 80%, 85%, 90%, 95%,97%, 98%, 99%, 99.5% or 100% identity with an amino acid sequence of awild type A2M polypeptide. In some embodiments, A2M variant polypeptidescan have an amino acid sequence sharing at least about 60%, 70%, 80%,85%, 90%, 95%, 97%, 98%, 99%, 99.5% or 100% identity with the amino acidsequence of a wild type A2M polypeptide.

Percent sequence identity can be calculated using computer programs ordirect sequence comparison. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D.W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The BLASTPand TBLASTN programs are publicly available from NCBI and other sources.The Smith Waterman algorithm can also be used to determine percentidentity. Exemplary parameters for amino acid sequence comparisoninclude the following: 1) algorithm from Needleman and Wunsch (J. Mol.Biol., 48:443-453 (1970)); 2) BLOSSUM62 comparison matrix from Hentikoffand Hentikoff (Proc. Nat. Acad. Sci. USA., 89:10915-10919 (1992)) 3) gappenalty=12; and 4) gap length penalty=4. A program useful with theseparameters can be publicly available as the “gap” program (GeneticsComputer Group, Madison, Wis.). The aforementioned parameters are thedefault parameters for polypeptide comparisons (with no penalty for endgaps). Alternatively, polypeptide sequence identity can be calculatedusing the following equation: % identity—(the number of identicalresidues)/(alignment length in amino acid residues)*100. For thiscalculation, alignment length includes internal gaps but does notinclude terminal gaps

Variant A2M polypeptides, or fragments thereof, include but are notlimited to, those containing as a primary amino acid sequence all orpart of one or more of the amino acid sequence encoded by SEQ ID NOs:6-83, and fragments of these proteins, including altered sequences inwhich functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a silent change. The variantA2M polypeptides can include all or part of the amino acid sequenceencoded by SEQ ID NO: 3. The variant A2M polypeptides can be, forexample, any number of between 4-20, 20-50, 50-100, 100-300, 300-600,600-1000, 1000-1450 consecutive amino acids containing one or more aminoacids sequences of SEQ ID NOs: 6-83. The variant A2M polypeptide can beless than or equal to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 250, 300,350, 400, 500, 600, 700, 800, 900, 1000, and 1450 amino acids in lengthand contain, as part of the sequence one or more sequences of SEQ IDNOs: 6-83. Variant A2M polypeptides includes polypeptide sequenceshaving at least 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%,70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60% sequenceidentity or similarity to any variant A2M polypeptide containing one ormore sequences of SEQ ID NOs: 6-83.

The variant A2M polypeptides provided herein also include proteinscharacterized by amino acid sequences similar to those of purifiedproteins but into which modification are naturally provided ordeliberately engineered. For example, modifications, in the variant A2Mpeptide or variant A2M DNA sequence, can be made by those skilled in theart using known techniques. Modifications of interest in the proteinsequences can include the alteration, substitution, replacement,insertion or deletion of a selected amino acid residue in the codingsequence. For example, one or more of the cysteine residues can bedeleted or replaced with another amino acid to alter the conformation ofthe molecule. Techniques for such alteration, substitution, replacement,insertion or deletion are well known to those skilled in the art (see,e.g., U.S. Pat. No. 4,518,584). Preferably, such alteration,substitution, replacement, insertion or deletion retains the desiredactivity of the protein. Regions of the protein that are important forthe protein function can be determined by various methods known in theart including the alanine-scanning method which involves systematicsubstitution of single or multiple amino acids with alanine, followed bytesting the resulting alanine-containing variant for biologicalactivity. This type of analysis can be used to determine the importanceof the substituted amino acid(s) in biological activity.

The bait region of A2M is a segment that is susceptible to proteolyticcleavage, and which, upon cleavage, initiates a conformational change inthe A2M molecule resulting in the collapse of the structure around theprotease. For the exemplary A2M sequences set forth in SEQ ID NO: 3, thebait region corresponds to amino acids 690-728. For the exemplary A2Msequences set forth in SEQ ID NO: 1 and 2, the bait region correspondsto the nucleotides encoding amino acids 690-728 (SEQ ID NO: 5).

A variant A2M polypeptide can comprise a bait region that is a variantof the bait region of wild-type A2M. For example, a bait region of avariant A2M polypeptide can be a mutant bait region, fragment of a baitregion, a bait region from another species, an isoform of a bait region,or a bait region containing multiple copies of one or more proteaserecognition sites and/or bait regions described herein, or anycombination thereof. A bait region of a variant A2M polypeptide caninclude a plurality of protease recognition sites arranged in series andcan be arranged in any order.

In some instances, a variant A2M polypeptide can comprise a sequencethat is substantially the same as wild type-A2M with respect to non-baitregions. For example, a variant A2M polypeptide can comprise a non-baitregion sequence that least about 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity to the correspondingnon-bait region sequence of wild-type A2M.

A bait region of a variant A2M polypeptide can have one or more variantbait regions comprising a sequence of at least about 60%, 70%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100% identity toany one of SEQ ID NOs: 6-30. A bait region of a variant A2M polypeptidecan have one or more consensus protease recognition sites. For example,a bait region of a variant A2M polypeptide can have 2 or more, or 3, 4,5, 6, 7, 8, 9, or 10 or more protease recognition sites. A bait regionof a variant A2M polypeptide can have one or more protease recognitionsites. A bait region of a variant A2M polypeptide can have one or moreprotease recognition sites comprising a sequence of at least about 60%,70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or100% identity to any one of SEQ ID NOs: 31-81. A bait region of avariant A2M polypeptide can have one or more protease recognition sitescomprising a sequence of SEQ ID NO: 82 or SEQ ID NO: 83. A bait regionof a variant A2M polypeptide can have one or more consensus proteaserecognition sites. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moreprotease recognition sites. Exemplary protease recognition sites are setforth in Table 2.

Protease recognition sites or substrate bait regions can be consensussequences for serine proteases, threonine proteases, cysteine proteases,aspartate proteases, metalloproteases, glutamic acid proteases, or anycombination thereof compared to a wild type A2M protein. A variant A2Mpolypeptide can be characterized by at least a 10% increased inhibitionof or more proteases (e.g., types of proteases). For example, a variantA2M can be characterized by at least a 15%, 20%, 30%, 40%, 50%, 60%,70,80%, 90%, 1090%, 120%, 140%, 150%, 160%, 180%, 200%, 220%, 240%,260%, 280%, 300%, 350%, 400%, 450%, or 500%, or higher enhancedinhibition of one or more serine proteases, threonine proteases,cysteine proteases, aspartate proteases, metalloproteases, glutamic acidproteases, or any combination thereof compared to a wild type A2Mprotein. A variant A2M polypeptide can be characterized by an enhancedspecific inhibition of serine proteases, threonine proteases, cysteineproteases, aspartate proteases, metalloproteases, glutamic acidproteases, or any combination thereof. For example, a variant A2M can becharacterized by at least a 15%, 20%, 30%, 40%, 50%, 60%, 70, 80%, 90%,1090%, 120%, 140%, 150%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, 300%,350%, 400%, 450%, or 500%, or higher enhanced specific inhibition ofserine proteases, threonine proteases, cysteine proteases, aspartateproteases, metalloproteases, glutamic acid proteases, or any combinationthereof compared to a wild type A2M protein. A variant A2M polypeptidecan be characterized by an enhanced nonspecific inhibition of serineproteases, threonine proteases, cysteine proteases, aspartate proteases,metalloproteases, glutamic acid proteases, or any combination thereofcompared to a wild type A2M protein. For example, a variant A2M can becharacterized by at least a 15%, 20%, 30%, 40%, 50%, 60%, 70, 80%, 90%,1090%, 120%, 140%, 150%, 160%, 180%, 200%, 220%, 240%, 260%, 280%, 300%,350%, 400%, 450%, or 500%, or higher enhanced nonspecific inhibition ofserine proteases, threonine proteases, cysteine proteases, aspartateproteases, metalloproteases, glutamic acid proteases, or any combinationthereof compared to a wild type A2M protein. Inhibition of proteaseactivity of exemplary variant A2M polypeptides can be seen in Table 3.Inhibition of protease activity of other exemplary variant A2Mpolypeptides can be seen in Tables 4a and 4b.

A bait region of a variant A2M polypeptide can have one or more mutantbase regions. For example, a bait region of a variant A2M polypeptidecan have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more mutant baseregions. A bait region of a variant A2M polypeptide can have one or morebait region fragments. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or morebait region fragments. A fragment of a bait region of a variant A2Mpolypeptide can be a fragment of one or more sequences of SEQ ID NOs:6-83.

A bait region of a variant A2M polypeptide can have one or more mutantamino acids that are different than those amino acids in a wild-type A2Mpolypeptide. For example, a bait region of a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more mutant amino acidsthat are different than those amino acids in a wild-type A2Mpolypeptide. A bait region of a variant A2M polypeptide can have one ormore mutant amino acid regions that are different than those regions ina wild-type A2M polypeptide. For example, a bait region of a variant A2Mpolypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moremutant amino acid regions that are different than those regions in awild-type A2M polypeptide. A mutant bait region of a variant A2Mpolypeptide can replace or substitute a bait region in a wild-type A2Mpolypeptide. A mutant bait region of a variant A2M polypeptide cancomprise one or more sequences of any of SEQ ID NOs: 6-83.

The A2M variant polypeptides provided herein also include A2M variantproteins characterized by conservative amino acid sequences. Isolated orpurified variant A2M polypeptides can have one or more amino acidresidues within the polypeptide that are substituted by another aminoacid of a similar polarity that acts as a functional equivalent,resulting in a silent alteration. Substitutes for an amino acid withinthe sequence can be selected from other members of the class to whichthe amino acid belongs. For example, the non-polar (hydrophobic) aminoacids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine, and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. The aromatic amino acidsinclude phenylalanine, tryptophan, and tyrosine.

A bait region of a variant A2M polypeptide can have one or more baitregion isoforms. For example, a bait region of a variant A2M polypeptidecan have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait regionisoforms. A bait region of a variant A2M polypeptide can have one ormore mutant or engineered bait regions. For example, a bait region of avariant A2M polypeptide can have 2 or more, or 3, 4, 5, 6, 7, 8, 9, or10 or more mutant or engineered bait regions.

A bait region of a variant A2M polypeptide can have one or more copiesof one or more bait regions. The one or more bait regions can be thesame bait regions (repeats), different bait regions, or any combinationthereof. For example, a bait region of a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more copies of one ormore bait regions, wherein the one or more bait regions can be the samebait regions (repeats), different bait regions, or any combinationthereof.

A variant A2M polypeptide can comprise one or more bait regions derivedfrom different organisms, different species of an organism, or acombination thereof. For example, a variant A2M polypeptide can have 2or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait regions derived fromdifferent organisms, different species of an organism, or a combinationthereof. One or more bait regions derived from different organisms canbe derived from one or more different organisms and not from differentspecies of an organism. For example, one or more modified bait regionscan be derived from 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or moredifferent organisms and not contain 2 or more bait regions derived fromdifferent species of an organism. One or more bait regions derived fromdifferent species of an organism can be derived from one or moredifferent species of an organism and not from different organisms. Forexample, one or more modified bait regions can be derived from 2 ormore, or 3, 4, 5, 6, 7, 8, 9, or 10 or more different species of anorganism and not contain 2 or more bait regions derived from differentorganism. The modified bait regions can be derived from any animal,insect, plant, bacteria, viral, yeast, fish, reptile, amphibian, orfungi. The modified bait regions can be derived from any animal with A2Mor homologous protein, such as pig, mouse, rat, rabbit, cat, dog, frog,monkey, horse or goat.

A variant A2M polypeptide can comprise one or more bait regions ofvariant A2M polypeptides. For example, a variant A2M polypeptide canhave 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more bait region ofvariant A2M polypeptides. One or more bait region of variant A2Mpolypeptides can be derived from one or more different species. Forexample, one or more bait regions of variant A2M polypeptides can bederived from 2 or more, or 3, 4, 5, 6, 7, 8, 9, or 10 or more differentspecies. The bait region of variant A2M polypeptides can be derived fromany animal, insect, plant, bacteria, viral, yeast, fish, reptile,amphibian, or fungi species.

A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from oneor more proteins other than A2M.

A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from A2M.A variant A2M polypeptide can have a plurality of protease recognitionsites that can be one or more protease substrate bait regions from oneor more non-natural protein sequences. The non-natural protein sequencescan comprise one or more protease recognition sites in series and canfunction as bait for proteases. A variant A2M polypeptide can have aplurality of protease recognition sites that can be one or more proteasesubstrate bait regions from or any of the combination of bait regionsdescribed herein. A variant A2M polypeptide can have any number ofprotease bait regions arranged in series. A variant A2M polypeptide canhave any number of protease bait regions from any species and can bearranged in series. One or more protease substrate bait regions from oneor more proteins other than A2M or from the one or more non-naturalprotein sequences can be a suicide inhibitor. For example, a variant A2Mpolypeptide can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, ormore suicide inhibitor bait regions. A suicide inhibitor can be operableto covalently attach a protease to A2M. Examples of known recognitionsequences for exemplary ADAMTSs and MMPs in human aggrecan are indicatedin Table 1. Dash shows location of proteolysis.

TABLE 1 Protease Aggrecan Cleavage Site Sequence ADAMTSs 370 NITEGE-ARGS377 (SEQ ID NO: 144) ADAMTSs 1540 TASELE-GRGTI 1550 (SEQ ID NO: 145)ADAMTSs 1709 TFKEEE-GLGSV 1719 (SEQ ID NO: 146) MMP-8 370 NITEGE-ARGS377(SEQ ID NO: 144) MMPs 336 VDIPEN-FFG 344 (SEQ ID NO: 147) MMP-3 374ARGS-V 378 (SEQ ID NO: 148) MMP-13 379 ILTVKP-IFEV 388 (SEQ ID NO: 149)

A variant A2M polypeptide can be characterized by at least about a 10%increase in protease inhibitory effectiveness compared to the proteaseinhibitory effectiveness of a wild type A2M protein. For example, avariant A2M polypeptide can be characterized by at least about a 20,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% increase in protease inhibitory effectiveness when comparedto the protease inhibitory effectiveness of a wild type A2M protein. Avariant A2M polypeptide can be characterized by an increase in proteaseinhibitory effectiveness compared to the protease inhibitoryeffectiveness of a wild type A2M protein. For example, a variant A2Mpolypeptide can be characterized by an 1.2, 1.2, 1.4, 1.5, 1.6, 1.7,1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000 times increase in protease inhibitoryeffectiveness compared to the protease inhibitory effectiveness of awild type A2M protein.

A variant A2M polypeptide can be characterized as having an increasedability to inhibit one or more proteases compared to a wild-type A2Mpolypeptide. A variant A2M polypeptide can have an ability to inhibitone or more proteases that is at least 1.5 times higher than the abilityof a wild-type A2M polypeptide to inhibit the one or more proteases. Forexample, a variant A2M polypeptide can have an ability to inhibit one ormore proteases that is at least 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 times higher than theability of a wild-type A2M polypeptide to inhibit the one or moreproteases. A variant A2M polypeptide can have an ability to inhibit oneor more proteases that is from 1.5-100 times higher than the ability ofa wild-type A2M polypeptide to inhibit the one or more proteases. Forexample, a variant A2M polypeptide can have an ability to inhibit one ormore proteases that is from 1.6-100, 1.7-100, 1.8-100, 1.9-100, 2-100,2.1-100, 2.2-100, 2.3-100, 2.4-100, 2.5-100, 2.6-100, 2.7-100, 2.8-100,2.9-100, 3.0-100, 3.1-100, 3.2-100, 3.3-100, 3.4-100, 3.5-100, 3.6-100,3.7-100, 3.8-100, 3.9-100, 4-100, 5-100, 6-100, 7-100, 8-100, 9-100,10-100, 11-100, 12-100, 13-100, 14-100, 15-100, 16-100, 17-100, 18-100,19-100, 20-100, 25-100, 30-100, 35-100, 40-100, 45-100, 50-100, 60-100,70-100, 80-100, 90-100, 1.5-90, 1.6-90, 1.7-90, 1.8-90, 1.9-90, 2-90,2.1-90, 2.2-90, 2.3-90, 2.4-90, 2.5-90, 2.6-90, 2.7-90, 2.8-90, 2.9-90,3.0-90, 3.1-90, 3.2-90, 3.3-90, 3.4-90, 3.5-90, 3.6-90, 3.7-90, 3.8-90,3.9-90, 4-90, 5-90, 6-90, 7-90, 8-90, 9-90, 10-90, 11-90, 12-90, 13-90,14-90, 15-90, 16-90, 17-90, 18-90, 19-90, 20-90, 25-90, 30-90, 35-90,40-90, 45-90, 50-90, 60-90, 70-90, 80-90, 1.5-80, 1.6-80, 1.7-80,1.8-80, 1.9-80, 2-80, 2.1-80, 2.2-80, 2.3-80, 2.4-80, 2.5-80, 2.6-80,2.7-80, 2.8-80, 2.9-80, 3.0-80, 3.1-80, 3.2-80, 3.3-80, 3.4-80, 3.5-80,3.6-80, 3.7-80, 3.8-80, 3.9-80, 4-80, 5-80, 6-80, 7-80, 8-80, 9-80,10-80, 11-80, 12-80, 13-80, 14-80, 15-80, 16-80, 17-80, 18-80, 19-80,20-80, 25-80, 30-80, 35-80, 40-80, 45-80, 50-80, 60-80, 70-80, 1.5-70,1.6-70, 1.7-70, 1.8-70, 1.9-70, 2-70, 2.1-70, 2.2-70, 2.3-70, 2.4-70,2.5-70, 2.6-70, 2.7-70, 2.8-70, 2.9-70, 3.0-70, 3.1-70, 3.2-70, 3.3-70,3.4-70, 3.5-70, 3.6-70, 3.7-70, 3.8-70, 3.9-70, 4-70, 5-70, 6-70, 7-70,8-70, 9-70, 10-70, 11-70, 12-70, 13-70, 14-70, 15-70, 16-70, 17-70,18-70, 19-70, 20-70, 25-70, 30-70, 35-70, 40-70, 45-70, 50-70, 60-70,1.5-60, 1.6-60, 1.7-60, 1.8-60, 1.9-60, 2-60, 2.1-60, 2.2-60, 2.3-60,2.4-60, 2.5-60, 2.6-60, 2.7-60, 2.8-60, 2.9-60, 3.0-60, 3.1-60, 3.2-60,3.3-60, 3.4-60, 3.5-60, 3.6-60, 3.7-60, 3.8-60, 3.9-60, 4-60, 5-60,6-60, 7-60, 8-60, 9-60, 10-60, 11-60, 12-60, 13-60, 14-60, 15-60, 16-60,17-60, 18-60, 19-60, 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60,1.5-50, 1.6-50, 1.7-50, 1.8-50, 1.9-50, 2-50, 2.1-50, 2.2-50, 2.3-50,2.4-50, 2.5-50, 2.6-50, 2.7-50, 2.8-50, 2.9-50, 3.0-50, 3.1-50, 3.2-50,3.3-50, 3.4-50, 3.5-50, 3.6-50, 3.7-50, 3.8-50, 3.9-50, 4-50, 5-50,6-50, 7-50, 8-50, 9-50, 10-50, 11-50, 12-50, 13-50, 14-50, 15-50, 16-50,17-50, 18-50, 19-50, 20-50, 25-50, 30-50, 35-50, 40-50, 1.5-40, 1.6-40,1.7-40, 1.8-40, 1.9-40, 2-40, 2.1-40, 2.2-40, 2.3-40, 2.4-40, 2.5-40,2.6-40, 2.7-40, 2.8-40, 2.9-40, 3.0-40, 3.1-40, 3.2-40, 3.3-40, 3.4-40,3.5-40, 3.6-40, 3.7-40, 3.8-40, 3.9-40, 4-40, 5-40, 6-40, 7-40, 8-40,9-40, 10-40, 11-40, 12-40, 13-40, 14-40, 15-40, 16-40, 17-40, 18-40,19-40, 20-40, 25-40, 30-40, 1.5-30, 1.6-30, 1.7-30, 1.8-30, 1.9-30,2-30, 2.1-30, 2.2-30, 2.3-30, 2.4-30, 2.5-30, 2.6-30, 2.7-30, 2.8-30,2.9-30, 3.0-30, 3.1-30, 3.2-30, 3.3-30, 3.4-30, 3.5-30, 3.6-30, 3.7-30,3.8-30, 3.9-30, 4-30, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 11-30, 12-30,13-30, 14-30, 15-30, 16-30, 17-30, 18-30, 19-30, 20-30, 1.5-20, 1.6-20,1.7-20, 1.8-20, 1.9-20, 2-20, 2.1-20, 2.2-20, 2.3-20, 2.4-20, 2.5-20,2.6-20, 2.7-20, 2.8-20, 2.9-20, 3.0-20, 3.1-20, 3.2-20, 3.3-20, 3.4-20,3.5-20, 3.6-20, 3.7-20, 3.8-20, 3.9-20, 4-20, 5-20, 6-20, 7-20, 8-20,9-20, 10-20, 11-20, 12-20, 13-20, 14-20, 15-20, 1.5-15, 1.6-15, 1.7-15,1.8-15, 1.9-15, 2-15, 2.1-15, 2.2-15, 2.3-15, 2.4-15, 2.5-15, 2.6-15,2.7-15, 2.8-15, 2.9-15, 3.0-15, 3.1-15, 3.2-15, 3.3-15, 3.4-15, 3.5-15,3.6-15, 3.7-15, 3.8-15, 3.9-15, 4-15, 5-15, 6-15, 7-15, 8-15, 9-15,10-15, 11-15, 12-15, 13-15, 14-15, 1.5-10, 1.6-10, 1.7-10, 1.8-10,1.9-10, 2-10, 2.1-10, 2.2-10, 2.3-10, 2.4-10, 2.5-10, 2.6-10, 2.7-10,2.8-10, 2.9-10, 3.0-10, 3.1-10, 3.2-10, 3.3-10, 3.4-10, 3.5-10, 3.6-10,3.7-10, 3.8-10, 3.9-10, 4-10, 5-10, 6-10, 7-10, 8-10, 9-10, 1.5-9,1.6-9, 1.7-9, 1.8-9, 1.9-9, 2-9, 2.1-9, 2.2-9, 2.3-9, 2.4-9, 2.5-9,2.6-9, 2.7-9, 2.8-9, 2.9-9, 3.0-9, 3.1-9, 3.2-9, 3.3-9, 3.4-9, 3.5-9,3.6-9, 3.7-9, 3.8-9, 3.9-9, 4-9, 5-9, 6-9, 7-9, 8-9, 1.5-8, 1.6-8,1.7-8, 1.8-8, 1.9-8, 2-8, 2.1-8, 2.2-8, 2.3-8, 2.4-8, 2.5-8, 2.6-8,2.7-8, 2.8-8, 2.9-8, 3.0-8, 3.1-8, 3.2-8, 3.3-8, 3.4-8, 3.5-8, 3.6-8,3.7-8, 3.8-8, 3.9-8, 4-8, 5-8, 6-8, 7-8, 1.5-7, 1.6-7, 1.7-7, 1.8-7,1.9-7, 2-7, 2.1-7, 2.2-7, 2.3-7, 2.4-7, 2.5-7, 2.6-7, 2.7-7, 2.8-7,2.9-7, 3.0-7, 3.1-7, 3.2-7, 3.3-7, 3.4-7, 3.5-7, 3.6-7, 3.7-7, 3.8-7,3.9-7, 4-7, 5-7, 6-7, 1.5-6, 1.6-6, 1.7-6, 1.8-6, 1.9-6, 2-6, 2.1-6,2.2-6, 2.3-6, 2.4-6, 2.5-6, 2.6-6, 2.7-6, 2.8-6, 2.9-6, 3.0-6, 3.1-6,3.2-6, 3.3-6, 3.4-6, 3.5-6, 3.6-6, 3.7-6, 3.8-6, 3.9-6, 4-6, 5-6, 1.5-5,1.6-5, 1.7-5, 1.8-5, 1.9-5, 2-5, 2.1-5, 2.2-5, 2.3-5, 2.4-5, 2.5-5,2.6-5, 2.7-5, 2.8-5, 2.9-5, 3.0-5, 3.1-5, 3.2-5, 3.3-5, 3.4-5, 3.5-5,3.6-5, 3.7-5, 3.8-5, 3.9-5, 4-5, 1.5-4, 1.6-4, 1.7-4, 1.8-4, 1.9-4, 2-4,2.1-4, 2.2-4, 2.3-4, 2.4-4, 2.5-4, 2.6-4, 2.7-4, 2.8-4, 2.9-4, 3.0-4,3.1-4, 3.2-4, 3.3-4, 3.4-4, 3.5-4, 3.6-4, 3.7-4, 3.8-4, 3.9-4, 1.5-3,1.6-3, 1.7-3, 1.8-3, 1.9-3, 2-3, 2.1-3, 2.2-3, 2.3-3, 2.4-3, 2.5-3,2.6-3, 2.7-3, 2.8-3, 2.9-3, 1.5-2, 1.6-2, 1.7-2, 1.8-2, or 1.9-2 timeshigher than the ability of a wild-type A2M polypeptide to inhibit theone or more proteases.

The one or more proteases can include a matrix metalloprotease, such asMMP1 (Interstitial collagenase), MMP2 (Gelatinase-A), MMP3 (Stromelysin1), MMP7 (Matrilysin, PUMP 1), MMP8 (Neutrophil collagenase), MMP9(Gelatinase-B), MMP10 (Stromelysin 2), MMP11 (Stromelysin 3), MMP12(Macrophage metalloelastase), MMP13 (Collagenase 3), MMP14 (MT1-MMP),MMP15 (MT2-MMP), MMP16 (MT3-MMP), MMP17 (MT4-MMP), MMP18 (Collagenase 4,xcol4, Xenopus collagenase), MMP19 (RASI-1, stromelysin-4), MMP20(Enamelysin), MMP21 (X-MMP), MMP23A (CA-MMP), MMP23B, MMP24 (MT5-MMP),MMP25 (MT6-MMP), MMP26 (Matrilysin-2, endometase), MMP27 (MMP-22,C-MMP), MMP28 (Epilysin); A Disintegrin and Metalloproteinase withThrombospondin Motifs protease, such as ADAMTS1, ADAMTS2, ADAMTS3,ADAMTS4, ADAMTS5 (ADAMTS11), ADAMTS6, ADAMTS7, ADAMTS8 (METH-2),ADAMTS9, ADAMTS10, ADAMTS12, ADAMTS13, ADAMTS14, ADAMTS15, ADAMTS16,ADAMTS17, ADAMTS18, ADAMTS19, ADAMTS20; chymotrypsin; trypsin; elastase;compliment factors; clotting factors; thrombin; plasmin; subtilisin;Neprilysin; Procollagen peptidase; Thermolysin; Pregnancy-associatedplasma protein A; Bone morphogenetic protein 1; Lysostaphin; Insulindegrading enzyme; ZMPSTE2; ZMPSTE4; ZMPSTE24; and acetylcholinesterase.

A variant A2M polypeptide can be characterized as having an increasedability to prevent FAC formation compared to a wild-type A2Mpolypeptide. A variant A2M polypeptide can have an ability to preventFAC formation that is at least 1.5 times higher than the ability of awild-type A2M polypeptide to prevent FAC formation. For example, avariant A2M polypeptide can have an ability to prevent FAC formationthat is at least 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, or 100 times higher than the ability of awild-type A2M polypeptide to prevent FAC formation. A variant A2Mpolypeptide can have an ability to prevent FAC formation that is from1.5-100 times higher than the ability of a wild-type A2M polypeptide toprevent FAC formation. For example, a variant A2M polypeptide can havean ability to prevent FAC formation that is from 1.6-100, 1.7-100,1.8-100, 1.9-100, 2-100, 2.1-100, 2.2-100, 2.3-100, 2.4-100, 2.5-100,2.6-100, 2.7-100, 2.8-100, 2.9-100, 3.0-100, 3.1-100, 3.2-100, 3.3-100,3.4-100, 3.5-100, 3.6-100, 3.7-100, 3.8-100, 3.9-100, 4-100, 5-100,6-100, 7-100, 8-100, 9-100, 10-100, 11-100, 12-100, 13-100, 14-100,15-100, 16-100, 17-100, 18-100, 19-100, 20-100, 25-100, 30-100, 35-100,40-100, 45-100, 50-100, 60-100, 70-100, 80-100, 90-100, 1.5-90, 1.6-90,1.7-90, 1.8-90, 1.9-90, 2-90, 2.1-90, 2.2-90, 2.3-90, 2.4-90, 2.5-90,2.6-90, 2.7-90, 2.8-90, 2.9-90, 3.0-90, 3.1-90, 3.2-90, 3.3-90, 3.4-90,3.5-90, 3.6-90, 3.7-90, 3.8-90, 3.9-90, 4-90, 5-90, 6-90, 7-90, 8-90,9-90, 10-90, 11-90, 12-90, 13-90, 14-90, 15-90, 16-90, 17-90, 18-90,19-90, 20-90, 25-90, 30-90, 35-90, 40-90, 45-90, 50-90, 60-90, 70-90,80-90, 1.5-80, 1.6-80, 1.7-80, 1.8-80, 1.9-80, 2-80, 2.1-80, 2.2-80,2.3-80, 2.4-80, 2.5-80, 2.6-80, 2.7-80, 2.8-80, 2.9-80, 3.0-80, 3.1-80,3.2-80, 3.3-80, 3.4-80, 3.5-80, 3.6-80, 3.7-80, 3.8-80, 3.9-80, 4-80,5-80, 6-80, 7-80, 8-80, 9-80, 10-80, 11-80, 12-80, 13-80, 14-80, 15-80,16-80, 17-80, 18-80, 19-80, 20-80, 25-80, 30-80, 35-80, 40-80, 45-80,50-80, 60-80, 70-80, 1.5-70, 1.6-70, 1.7-70, 1.8-70, 1.9-70, 2-70,2.1-70, 2.2-70, 2.3-70, 2.4-70, 2.5-70, 2.6-70, 2.7-70, 2.8-70, 2.9-70,3.0-70, 3.1-70, 3.2-70, 3.3-70, 3.4-70, 3.5-70, 3.6-70, 3.7-70, 3.8-70,3.9-70, 4-70, 5-70, 6-70, 7-70, 8-70, 9-70, 10-70, 11-70, 12-70, 13-70,14-70, 15-70, 16-70, 17-70, 18-70, 19-70, 20-70, 25-70, 30-70, 35-70,40-70, 45-70, 50-70, 60-70, 1.5-60, 1.6-60, 1.7-60, 1.8-60, 1.9-60,2-60, 2.1-60, 2.2-60, 2.3-60, 2.4-60, 2.5-60, 2.6-60, 2.7-60, 2.8-60,2.9-60, 3.0-60, 3.1-60, 3.2-60, 3.3-60, 3.4-60, 3.5-60, 3.6-60, 3.7-60,3.8-60, 3.9-60, 4-60, 5-60, 6-60, 7-60, 8-60, 9-60, 10-60, 11-60, 12-60,13-60, 14-60, 15-60, 16-60, 17-60, 18-60, 19-60, 20-60, 25-60, 30-60,35-60, 40-60, 45-60, 50-60, 1.5-50, 1.6-50, 1.7-50, 1.8-50, 1.9-50,2-50, 2.1-50, 2.2-50, 2.3-50, 2.4-50, 2.5-50, 2.6-50, 2.7-50, 2.8-50,2.9-50, 3.0-50, 3.1-50, 3.2-50, 3.3-50, 3.4-50, 3.5-50, 3.6-50, 3.7-50,3.8-50, 3.9-50, 4-50, 5-50, 6-50, 7-50, 8-50, 9-50, 10-50, 11-50, 12-50,13-50, 14-50, 15-50, 16-50, 17-50, 18-50, 19-50, 20-50, 25-50, 30-50,35-50, 40-50, 1.5-40, 1.6-40, 1.7-40, 1.8-40, 1.9-40, 2-40, 2.1-40,2.2-40, 2.3-40, 2.4-40, 2.5-40, 2.6-40, 2.7-40, 2.8-40, 2.9-40, 3.0-40,3.1-40, 3.2-40, 3.3-40, 3.4-40, 3.5-40, 3.6-40, 3.7-40, 3.8-40, 3.9-40,4-40, 5-40, 6-40, 7-40, 8-40, 9-40, 10-40, 11-40, 12-40, 13-40, 14-40,15-40, 16-40, 17-40, 18-40, 19-40, 20-40, 25-40, 30-40, 1.5-30, 1.6-30,1.7-30, 1.8-30, 1.9-30, 2-30, 2.1-30, 2.2-30, 2.3-30, 2.4-30, 2.5-30,2.6-30, 2.7-30, 2.8-30, 2.9-30, 3.0-30, 3.1-30, 3.2-30, 3.3-30, 3.4-30,3.5-30, 3.6-30, 3.7-30, 3.8-30, 3.9-30, 4-30, 5-30, 6-30, 7-30, 8-30,9-30, 10-30, 11-30, 12-30, 13-30, 14-30, 15-30, 16-30, 17-30, 18-30,19-30, 20-30, 1.5-20, 1.6-20, 1.7-20, 1.8-20, 1.9-20, 2-20, 2.1-20,2.2-20, 2.3-20, 2.4-20, 2.5-20, 2.6-20, 2.7-20, 2.8-20, 2.9-20, 3.0-20,3.1-20, 3.2-20, 3.3-20, 3.4-20, 3.5-20, 3.6-20, 3.7-20, 3.8-20, 3.9-20,4-20, 5-20, 6-20, 7-20, 8-20, 9-20, 10-20, 11-20, 12-20, 13-20, 14-20,15-20, 1.5-15, 1.6-15, 1.7-15, 1.8-15, 1.9-15, 2-15, 2.1-15, 2.2-15,2.3-15, 2.4-15, 2.5-15, 2.6-15, 2.7-15, 2.8-15, 2.9-15, 3.0-15, 3.1-15,3.2-15, 3.3-15, 3.4-15, 3.5-15, 3.6-15, 3.7-15, 3.8-15, 3.9-15, 4-15,5-15, 6-15, 7-15, 8-15, 9-15, 10-15, 11-15, 12-15, 13-15, 14-15, 1.5-10,1.6-10, 1.7-10, 1.8-10, 1.9-10, 2-10, 2.1-10, 2.2-10, 2.3-10, 2.4-10,2.5-10, 2.6-10, 2.7-10, 2.8-10, 2.9-10, 3.0-10, 3.1-10, 3.2-10, 3.3-10,3.4-10, 3.5-10, 3.6-10, 3.7-10, 3.8-10, 3.9-10, 4-10, 5-10, 6-10, 7-10,8-10, 9-10, 1.5-9, 1.6-9, 1.7-9, 1.8-9, 1.9-9, 2-9, 2.1-9, 2.2-9, 2.3-9,2.4-9, 2.5-9, 2.6-9, 2.7-9, 2.8-9, 2.9-9, 3.0-9, 3.1-9, 3.2-9, 3.3-9,3.4-9, 3.5-9, 3.6-9, 3.7-9, 3.8-9, 3.9-9, 4-9, 5-9, 6-9, 7-9, 8-9,1.5-8, 1.6-8, 1.7-8, 1.8-8, 1.9-8, 2-8, 2.1-8, 2.2-8, 2.3-8, 2.4-8,2.5-8, 2.6- 8, 2.7-8, 2.8-8, 2.9-8, 3.0-8, 3.1-8, 3.2-8, 3.3-8, 3.4-8,3.5-8, 3.6-8, 3.7-8, 3.8-8, 3.9-8, 4-8, 5-8, 6-8, 7-8, 1.5-7, 1.6-7,1.7-7, 1.8-7, 1.9-7, 2-7, 2.1-7, 2.2-7, 2.3-7, 2.4-7, 2.5-7, 2.6-7,2.7-7, 2.8-7, 2.9-7, 3.0-7, 3.1-7, 3.2-7, 3.3-7, 3.4-7, 3.5-7, 3.6-7,3.7-7, 3.8-7, 3.9-7, 4-7, 5-7, 6-7, 1.5-6, 1.6-6, 1.7-6, 1.8-6, 1.9-6,2-6, 2.1-6, 2.2-6, 2.3-6, 2.4-6, 2.5-6, 2.6-6, 2.7-6, 2.8-6, 2.9-6,3.0-6, 3.1-6, 3.2-6, 3.3-6, 3.4-6, 3.5-6, 3.6-6, 3.7-6, 3.8-6, 3.9-6,4-6, 5-6, 1.5-5, 1.6-5, 1.7-5, 1.8-5, 1.9-5, 2-5, 2.1-5, 2.2-5, 2.3-5,2.4-5, 2.5-5, 2.6-5, 2.7-5, 2.8-5, 2.9-5, 3.0-5, 3.1-5, 3.2-5, 3.3-5,3.4-5, 3.5-5, 3.6-5, 3.7-5, 3.8-5, 3.9-5, 4-5, 1.5-4, 1.6-4, 1.7-4,1.8-4, 1.9-4, 2-4, 2.1-4, 2.2-4, 2.3-4, 2.4-4, 2.5-4, 2.6-4, 2.7-4,2.8-4, 2.9-4, 3.0-4, 3.1-4, 3.2-4, 3.3-4, 3.4-4, 3.5-4, 3.6-4, 3.7-4,3.8-4, 3.9-4, 1.5-3, 1.6-3, 1.7-3, 1.8-3, 1.9-3, 2-3, 2.1-3, 2.2-3,2.3-3, 2.4-3, 2.5-3, 2.6-3, 2.7-3, 2.8-3, 2.9-3, 1.5-2, 1.6-2, 1.7-2,1.8-2, or 1.9-2 times higher than the ability of a wild-type A2Mpolypeptide to prevent FAC formation.

One aspect of the invention is a method for determining the enhancedinhibition of a protease by a variant A2M polypeptide comprising: a)providing a variant A2M polypeptide comprising a sequence of one or moreof SEQ ID NOs 6-83; b) contacting the variant A2M polypeptide with theprotease and a substrate cleaved by the protease; c) contacting awild-type A2M polypeptide with the protease and the substrate cleaved bythe protease; and d) comparing the amount of cleavage of the substratefrom step b) to the amount of cleavage of the substrate from step c),thereby determining the enhanced inhibition of the protease by thevariant A2M polypeptide.

Enzymatic glycoconjugation reactions can be targeted to glycosylationsites and to residues that are attached to glycosylation sites. Thetargeted glycosylation sites can be sites native to a wild-type A2Mprotein, native to a variant A2M polypeptide or, alternatively, they canbe introduced into a wild-type A2M or variant A2M polypeptide bymutation. Thus, a method for increasing the in vivo half-life of avariant A2M polypeptide is provided by the methods of the invention.

A variant A2M polypeptide can include an amino acid sequence thatmutated to insert, remove or relocate one or more glycosylation site inthe protein. When a site is added or relocated, it is not present or notpresent in a selected location in the wild-type A2M peptide. The mutantglycosylation site can be a point of attachment for a modified glycosylresidue that can be enzymatically conjugated to the glycosylation site.Using the methods of the invention, the glycosylation site can beshifted to any efficacious position on the peptide. For example, if thenative glycosylation site is sufficiently proximate or within the baitregion of variant A2M polypeptide peptide that conjugation interfereswith the ability to bind a protease, inhibit a protease, or acombination thereof, it is within the scope of the invention to engineera variant A2M polypeptide that includes a glycosylation site as modifiedor removed from the bait as necessary to provide a biologically activevariant A2M polypeptide.

Any glycosyltransferase or method of their use known in the art can beused for in vitro enzymatic synthesis of variant A2M polypeptides withcustom designed glycosylation patterns, various glycosyl structures, ora combination thereof possible. See, for example, U.S. Pat. Nos.5,876,980; 6,030,815; 5,728,554; 5,922,577; and WO/9831826;US2003180835; and WO 03/031464.

A variant A2M polypeptide can comprise one or more consensus sequencesfor a protease. A variant A2M polypeptide can comprise one or moreprotease recognition sites/sequences in Table 2.

TABLE 2 Exemplary variant A2M protease recognition sequences of bait regions for indicated protease Protease Recognition/Type Protease Cleavage Site Sequence Aggrecan ADAMTSTAQEAGEG (SEQ ID NO: 31), VSQELGQR (SEQ ID NO: 32) cleavage MMPIPENFFGV (SEQ ID NO: 33), SEDLVVQI (SEQ ID NO: 34), EAIPMSIPT (SEQ ID NO: 35)sites General or ADAMTS generalELEGRG (SEQ ID NO: 36), EEEGLG (SEQ ID NO: 37), EEEGGG (SEQ ID NO: 38),Multiple EXE-θ₄XG  ESESEG (SEQ ID NO: 39), EFEVEG (SEQ ID NO: 40), EIEEGG (SEQ ID NO: 41), cleavage (SEQ ID NO: 83)ERESTG (SEQ ID NO: 42), EREAQG (SEQ ID NO: 43), EKETGG (SEQ ID NO: 44),sites where θ is G, V,EREAQG (SEQ ID NO: 45), ETEGRG (SEQ ID NO: 46), ENEAGG (SEQ ID NO: 47),E, A, T, S, Q, P,EPESSG (SEQ ID NO: 48), EPESSG (SEQ ID NO: 49), ESESEG (SEQ ID NO: 50), N, or DEGEQEG (SEQ ID NO: 51), EPEPEG (SEQ ID NO: 52), EREAQG (SEQ ID NO: 53), where X is any EAEGTG (SEQ ID NO: 54), EFPEVEG (SEQ ID NO: 55)amino acid MMP general (G/P/E)XX(G/E)-GEEGVEEG (SEQ ID NO: 56), GARGLEG (SEQ ID NO: 57), GPPGLAPG (SEQ ID NO: 58),ΦXXGGYPGSSRG (SEQ ID NO: 59), GFAGLPNG (SEQ ID NO: 60), GGGGSLLG (SEQ ID NO: 61),(SEQ ID NO: 82)GPAGAARG (SEQ ID NO: 62), GLEGGGGG (SEQ ID NO: 63), GGGGSLLG (SEQ ID NO: 64),where Φ is G, V,GFFGFPIG (SEQ ID NO: 65), EPAGAARG (SEQ ID NO: 66), GDRGLPIG (SEQ ID NO: 67),L, S, A, F, or TGEPEGAKG (SEQ ID NO: 68), GFKEGVEG (SEQ ID NO: 69), GVEGVELG (SEQ ID NO: 70),where X is anyGFKEGVEG (SEQ ID NO: 71), GERGVLG (SEQ ID NO: 72), GGGSLLG (SEQ ID NO: 73),amino acidPEEGVEEG (SEQ ID NO: 74), GFKEGVEG (SEQ ID NO: 75), GFKEGVEG (SEQ ID NO: 76),GEPEGAKG (SEQ ID NO: 77) MMP and ADAMTS TEGEARGS (SEQ ID NO: 78)cleavage Consensus ADAMTS EGEGEGEG (SEQ ID NO: 79) Sequences ADAMTS-4EFRGVT (SEQ ID NO: 80) MMP-2 PRYLTA (SEQ ID NO: 81)

TABLE 3 Variant A2M protease inhibition (% of wild-type) for indicatedprotease Protease Inhibition (% of WT) Bait Region AggrecanasesCollagenases Gelatinases Stromolysin Inflammatory Proteases MatrilysinSEQ ID NO: ADAMTS4 ADAMTS5 MMP1 MMP8 MMP13 MMP2 MMP9 MMP3 ElastaseCathepsin G MMP7 6 7 222 79 117 81 145 58 39 127 131 157 147 8 120 97 4828 56 33 9 <100 39 12 64 9 124 145 67 33 88 27 20 27 93 66 80 10 159 412111 38 128 25 16 27 106 134 191 11 12 208 440 108 54 72 16 0 182 117 105160 13 290 194 91 81 76 65 71 189 97 54 128 14 236 105 60 39 66 10 14169 67 99 61 15 83 183 116 77 143 59 41 40 115 139 157 16 50-150 305 10192 104 92 84 180 91 105 105 17 18  0-100 154 91 82 117 58 53 38 86 83102 19  0-330 530 100 51 89 36 39 140 115 90 137 20 201 246 119 64 10037 41 122 124 117 170 21 68 217 84 39 46 66 45 26 59 58 94 22 104 316 6854 93 42 30 265 92 78 75 23 176 200 133 132 133 80 71 111 147 145 191 2425 144 376 101 36 138 43 25 86 100 117 140 26 27 50-150 114 109 43 54 1613 33 79 161 138 28 180 398 67 38 90 36 14 129 75 69 94 29 100-390  8587 29 70 16 5 101 98 84 104 30 93 296 67 29 96 3 18 <100 55 104 91

TABLE 4a Variant A2M protease inhibition (% of wild-type) of indicatedprotease Protease Inhibition (% of WT) Protease Mixtures Misc SerineProteases Bait Region Inflammatory Proteases IGD Collagen Chymo-Matrilysin SEQ ID NO Elastase Cathepsin G Substrate Substrate Trypsintrypsin MMP-7 84 75 100 71 85 86 87 88 89 90 550 100 80 91 92 93 94 9596 50 100 97 98 99 100 101 102 103 104 105 106 107 108 50 100 100 109110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 75 250 237125 75 250 150 126 105 311 229 250 86 50 127 102 324 229 180 84 25 128100 225 125 129 75 160 130 103 311 250 100 131 300 200 150 132 300 437243 120 20 10 133 350 100 75 134 300 250 220 135 300 100 140 136 250 376215 170 221 25 137 100 450 140 138 250 100 210 139 200 200 190 140 14175 100 142 200 300 140 143 250 350 110

TABLE 4b Variant A2M protease inhibition (% of wild-type) for indicatedprotease Protease Inhibition (% of WT) Bait Region AggrecanasesCollagenases Gelatinases Stromolysin Metalloelastase SEQ ID NO ADAMTS4ADAMTS5 MMP1 MMP8 MMP13 MMP2 MMP9 MMP3 MMP12 84 70 250 180 350 720 12060 170 100 85 78 174 170 86 91 74 120 87 160 170 150 40 150 100 70 40 8868 108 156 89 101 485 18 90 70 350 50 110 700 110 100 110 100 91 92 210250 170 160 90 110 50 80 93 27 66 119 94 121 395 173 95 153 272 79 96250 350 100 50 200 100 70 80 100 97 131 128 116 98 124 82 148 99 63 8764 100 50 400 40 20 210 110 50 50 101 168 97 124 102 187 354 127 103 168309 52 104 100 99 80 105 94 115 164 106 104 93 16 107 92 108 136 108 5090 390 40 350 110 70 330 109 112 105 122 110 101 208 51 111 105 89 113112 108 199 56 113 115 92 27 114 118 237 75 115 108 163 78 116 107 92116 117 100 190 11 118 119 100 170 90 120 88 100 121 121 64 68 115 122123 86 126 122 124 125 200 300 20 300 300 126 115 480 379 163 117 61 500130 132 127 82 205 327 210 131 118 500 130 170 128 400 20 129 200 20 130110 211 283 180 146 109 100 124 131 300 132 86 185 388 161 117 67 500100 136 133 250 134 200 200 135 300 136 80 373 370 133 113 47 500 130150 137 138 200 139 400 140 141 142 350 143

The present invention provides methods of improving or lengthening thein vivo half-lives of variant A2M polypeptides by conjugating awater-soluble polymer to the variant A2M polypeptides through an intactglycosyl linking group. In an exemplary embodiment, covalent attachmentof polymers, such as polyethylene glycol (PEG), to such variant A2Mpolypeptides affords variant A2M polypeptides having in vivo residencetimes, and pharmacokinetic and pharmacodynamic properties, enhancedrelative to the unconjugated variant A2M polypeptide.

The polymer backbone of the water-soluble polymer can be poly(ethyleneglycol) (PEG). However, it should be understood that other relatedpolymers are also suitable for use in the practice of this invention andthat the use of the term PEG or poly(ethylene glycol) is intended to beinclusive and not exclusive in this respect. The term PEG includespoly(ethylene glycol) in any of its forms, including alkoxy PEG,difunctional PEG, multiarmed PEG, forked PEG, branched PEG, pendent PEG(i.e. PEG or related polymers having one or more functional groupspendent to the polymer backbone), or PEG with degradable linkagestherein. The polymer backbone can be linear or branched. Branchedpolymer backbones are generally known in the art. Typically, a branchedpolymer has a central branch core moiety and a plurality of linearpolymer chains linked to the central branch core. PEG is commonly usedin branched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, pentaerythritol and sorbitol. Thecentral branch moiety can also be derived from several amino acids, suchas lysine. The branched poly(ethylene glycol) can be represented ingeneral form as R(PEG-OH)_(n) in which R represents the core moiety,such as glycerol or pentaerythritol, and n represents the number ofarms. Many other polymers are also suitable for the invention. Examplesof suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers of ethylene glycol and propylene glycol and the like,poly(oxyethylated polyol), poly(olefinic alcohol),polyvinylpyrrolidone), poly(hydroxypropylmethacrylamide), poly(α-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine) and copolymers, terpolymers, and mixturesthereof. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 100 Da toabout 100,000 Da often from about 6,000 Da to about 80,000 Da.

A variant A2M polypeptide can further comprise PEG. A variant A2Mpolypeptide can have one or more mutant or modified glycosylation sites.The modified glycosylation sites can comprise PEG. For example, avariant A2M polypeptide can have 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or more mutant or modified glycosylation sites. The conjugationor addition of PEG to a variant A2M polypeptide with one or moremodified or abnormal glycosylation sites can result in a variant A2Mpolypeptide with a longer half-life than the half-life of a wild-typeA2M protein without PEG when disposed within a subject, such as a jointor spine disc of a subject. The conjugation or addition of PEG to avariant A2M polypeptide with one or more modified or abnormalglycosylation sites can result in a variant A2M polypeptide with alonger half-life than the half-life of a variant A2M polypeptide withoutone or more modified glycosylation sites without PEG when disposedwithin a subject, such as a joint or spine disc of a subject. Theconjugation or addition of PEG to a variant A2M polypeptide with one ormore modified or abnormal glycosylation sites can result in a variantA2M polypeptide with a longer half-life than the half-life of a variantA2M polypeptide with one or more modified glycosylation sites withoutPEG when disposed within a subject, such as a joint or spine disc of asubject. For example, a variant A2M polypeptide with one or moremodified or abnormal glycosylation sites with PEG can have half-lifethat is 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6,6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000 times the half-life of awild type A2M protein without PEG, a variant A2M polypeptide with one ormore modified glycosylation sites without PEG, or a variant A2Mpolypeptide without one or more modified glycosylation sites withoutPEG. For example, a variant A2M polypeptide with one or more modified orabnormal glycosylation sites with PEG can have half-life that is 2 timesthe half-life of a wild type A2M protein composition with one or moremodified or abnormal glycosylation sites without PEG when disposedwithin a joint or spine disc of a subject.

The present invention further provides isolated polypeptides encoded bythe nucleic acid fragments of the present invention or by degeneratevariants of the nucleic acid fragments of the present invention. By“degenerate variant” can be intended nucleotide fragments which differfrom a nucleic acid fragment of the present invention (e.g., an ORF) bynucleotide sequence but, due to the degeneracy of the genetic code,encode an identical polypeptide sequence. Preferred nucleic acidfragments of the present invention are the ORFs that encode proteins.

Fragments of the A2M variants of the present invention which are capableof exhibiting biological activity are also encompassed by the presentinvention. Fragments of the A2M variants can be in linear form or theycan be cyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments can be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites. The presentinvention also provides both full-length and mature forms (for example,without a signal sequence or precursor sequence) of the disclosed A2Mvariants. The protein coding sequence can be identified in the sequencelisting by translation of the disclosed nucleotide sequences. The matureform of such A2M variants can be obtained by expression of a full-lengthpolynucleotide in a suitable mammalian cell or other host cell. Thesequence of the mature form of the A2M variants can be also determinablefrom the amino acid sequence of the full-length form. Where A2M variantsof the present invention are membrane bound, soluble forms of the A2Mvariants are also provided. In such forms, part or all of the regionscausing the A2M variants to be membrane bound are deleted so that theA2M variants are fully secreted from the cell in which it can beexpressed. A2M variant compositions of the present invention can furthercomprise an acceptable carrier, such as a hydrophilic, e.g.,pharmaceutically acceptable, carrier.

Variant A2M Polynucleotide Compositions

As used herein, “A2M polynucleotide,” when used with reference to SEQ IDNOs: 1 or 2, means the polynucleotide sequence of SEQ ID NO: 1 or 2, orfragments thereof, as well as any nucleic acid variants which includeone or more insertions, deletions, mutations, or a combination thereof.The insertions, deletions, and mutations are preferably within thepolynucleotide sequence encoding the bait region of the A2M protein.Similarly, “A2M cDNA”, “A2M coding sequence” or “A2M coding nucleicacid”, when used with reference to SEQ ID NOs: 1 or 2, means the nucleicacid sequences of SEQ ID NOs: 1 or 2, or fragments thereof, as well asnucleic acid variants which include one or more mutations, insertions,deletions, or a combination thereof. The A2M polynucleotides, orfragments thereof, can be manipulated using conventional techniques inmolecular biology so as to create variant A2M recombinant polynucleotideconstructs, encoding the variant A2M polypeptides that express variantA2M polypeptides. Variant A2M polynucleotides include nucleotidesequences having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,90%, 89%, 88%, 87%, 86%, or 85% sequence identity to SEQ ID NOs: 1 and2. A2M coding sequences includes nucleotide sequences having at least99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, or85% sequence identity to any one of SEQ ID NOs: 1 and 2.

In one aspect, provided herein is a variant A2M polynucleotidenucleotide composition. Numerous polynucleotide sequences encodingwild-type A2M proteins from various organisms have been determined. AnyA2M DNA sequence identified can be subsequently obtained by chemicalsynthesis and/or a polymerase chain reaction (PCR) technique such asoverlap extension method. For a short sequence, completely de novosynthesis may be sufficient; whereas further isolation of full lengthcoding sequence from a human cDNA or genomic library using a syntheticprobe may be necessary to obtain a larger gene. Alternatively, a nucleicacid sequence encoding an A2M polypeptide can be isolated from a humancDNA or genomic DNA library using standard cloning techniques such aspolymerase chain reaction (PCR), where homology-based primers can oftenbe derived from a known nucleic acid sequence encoding an A2Mpolypeptide.

cDNA libraries suitable for obtaining a coding sequence for a wild-typeA2M polypeptide can be obtained commercially or can be constructed. Thegeneral methods of isolating mRNA, making cDNA by reverse transcription,ligating cDNA into a recombinant vector, transfecting into a recombinanthost for propagation, screening, and cloning are well known. Uponobtaining an amplified segment of nucleotide sequence by PCR, thesegment can be further used as a probe to isolate the full-lengthpolynucleotide sequence encoding the wild-type A2M protein from the cDNAlibrary. A similar procedure can be followed to obtain a full lengthsequence encoding a wild-type A2M protein from a human genomic library.Human genomic libraries are commercially available or can be constructedaccording to various art-recognized methods. In general, to construct agenomic library, the DNA is first extracted from a tissue where apeptide is likely found. The DNA is then either mechanically sheared orenzymatically digested to yield fragments of about 12-20 kb in length.The fragments are subsequently separated by gradient centrifugation frompolynucleotide fragments of undesired sizes and are inserted inbacteriophage λ vectors. These vectors and phages are packaged in vitro.Recombinant phages are analyzed by plaque hybridization.

Based on sequence homology, degenerate oligonucleotides can be designedas primer sets and PCR can be performed under suitable conditions toamplify a segment of nucleotide sequence from a cDNA or genomic library.Using the amplified segment as a probe, the full-length nucleic acidencoding a wild-type A2M protein can be obtained

Upon acquiring a nucleic acid sequence encoding a wild-type A2M protein,the coding sequence can be subcloned into a vector, for instance, anexpression vector, so that a recombinant wild-type A2M protein can beexpressed mutated into a variant A2M polypeptide of the inventionproduced from the resulting construct. Further modifications to thewild-type A2M protein coding sequence, for example, nucleotidesubstitutions, may be subsequently made to alter the bait region of theA2M protein.

The present invention further provides isolated polypeptides encoded bythe polynucleotides, or fragments thereof, of the present invention orby degenerate variants of the polynucleotides, or fragments thereof, ofthe present invention. Preferred polynucleotides, or fragments thereof,of the present invention are the ORFs that encode A2M variants.

A variant A2M polynucleotide can be made by mutating the polynucleotidesequence encoding a wild-type A2M protein. This can be achieved by usingany known mutagenesis methods. Exemplary modifications to a wild-typeA2M polynucleotide for accepting variant bait regions described hereininclude those in SEQ ID NO 2. Exemplary modifications to an A2Mnucleotide include inserting or substituting a nucleotide sequenceencoding a variant bait region of SEQ ID NOs: 6-30 or a variant baitregion comprising one or more protease recognition sequences of SEQ IDNOs 31-83, into the wild-type A2M polynucleotide sequence of SEQ ID NO:1 and the variant A2M acceptor polynucleotide sequence of SEQ ID NO 2.Mutagenesis procedures can be used separately or in combination toproduce variants of a set of nucleic acids, and hence variants ofencoded polypeptides. Kits for mutagenesis are commercially available.

In one aspect, provided herein are methods of making any of the variantA2M polynucleotides. A method of making a variant A2M polynucleotide cancomprise inserting or substituting a variant bait region into awild-type A2M polynucleotide sequence or substantially similar sequence.The substantially similar sequence can be SEQ ID NO 2. One aspect of theinvention is a method for making a variant A2M polynucleotidecomprising: a) providing a vector containing a variant A2Mpolynucleotide comprising a sequence of SEQ ID NO 2; b) digesting thevector containing a variant A2M polynucleotide with restrictionendonucleases to form a linear vector; c) ligating one end of the one ormore polynucleotides encoding one or more of the variant bait regions ofSEQ ID NOs: 6-30 or variant bait regions comprising one or more proteaserecognition sequences of SEQ ID NOs 31-83 to one end of the linearvector; and d) ligating the other end of the one or more polynucleotidesencoding one or more of the variant bait regions of SEQ ID NOs: 6-30 orthe variant bait regions comprising one or more protease recognitionsequences of SEQ ID NOs 31-83 to the other end of the linear vector,thereby forming a vector containing a variant A2M polynucleotidecomprising the variant bait regions of SEQ ID NOs: 6-30 or variant baitregions comprising one or more protease recognition sequences of SEQ IDNOs 31-83.

Protein Production

A variety of methodologies known in the art can be utilized to obtainany one of the isolated A2M variant proteins of the present invention.At the simplest level, the amino acid sequence can be synthesized usingcommercially available peptide synthesizers. Such polypeptides can besynthesized with or without a methionine on the amino terminus.Chemically synthesized polypeptides can be oxidized using methods setforth in these references to form disulfide bridges. The syntheticallyconstructed A2M variant sequences, by virtue of sharing primary,secondary or tertiary structural and/or conformational characteristicswith A2M variants can possess biological properties in common therewith,including protease inhibitory activity. This technique can beparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the A2M variants. Thus, they can be employed asbiologically active or immunological substitutes for natural, purifiedA2M variants in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies.

The A2M variant polypeptides of the present invention can alternativelybe purified from cells which have been altered to express the desiredA2M variant. As used herein, a cell can be said to be altered to expressa desired A2M variant polypeptide or protein when the cell, throughgenetic manipulation, is made to produce an A2M variant polypeptidewhich it normally does not produce or which the cell normally producesat a lower level. One skilled in the art can readily adapt proceduresfor introducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the A2M variant polypeptides of the present invention.

A variant A2M polypeptide can be a recombinant protein, or fragmentsthereof, and can be produced in a host cell or in vitro system.Recombinant polypeptides and protein promoters can be inserted in such amanner that it can be operatively produced in a host cell, for example,a bacterial culture or lower eukaryotes such as yeast or insects or inprokaryotes or any host know in the art. A variant A2M recombinantprotein can be produced in a bacterium, yeast, fungi, insect, ormammalian host cell, or a cell free system. For example, a variant A2Mpolypeptide can be produced in Escherichia coli, Bacillus subtilis,Salmonella typhimurium, Corynebacterium, Saccharomyces cerevisiae,Schizosaccharomyces pombe Kluyveromyces strains, Candida, Pichiapastoris, baculovirus-infected insect cells, or mammalian cells such asCOS cells, BHK cells, 293 cells, 3T3 cells, NS0 hybridoma cells, babyhamster kidney (BHK) cells, PER.C6™ human cells, HEK293 cells orCricetulus griseus (CHO) cells. A variant A2M polypeptide can beproduced by transient expression, stable cell lines, BacMam-mediatedtransient transduction, or cell-free protein production.

The variant A2M polypeptides can also be produced by operably linkingthe isolated variant A2M polynucleotides to suitable control sequencesin one or more insect expression vectors, and employing an insectexpression system. Materials and methods for baculovirus/insect cellexpression systems are commercially available in kit form from, e.g.,Invitrogen, San Diego, Calif., U.S.A. (the MaxBat™ kit), and suchmethods are well known in the art, as described in Summers and Smith,Tex. Agricultural Experiment Station Bulletin No. 1555 (1987),incorporated herein by reference.

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the variant A2M nucleotide sequence of interest can be ligatedto an adenovirus transcription/translation control complex, for example,the late promoter and tripartite leader sequence. This chimeric gene canthen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genomecan result in a recombinant virus that is viable and capable ofexpressing the variant A2M gene product in infected hosts. Specificinitiation signals can also be required for efficient translation ofinserted nucleotide sequences. These signals include the ATG initiationcodon and adjacent sequences. In cases where an entire variant A2M geneor cDNA, including its own initiation codon and adjacent sequences, isinserted into the appropriate expression vector, for example, a pJ608mammalian expression vector (FIG. 23) no additional translationalcontrol signals are needed. Exogenous translational control signals,such as the ATG initiation codon, can be provided.

Host cells can be genetically engineered to contain the variant A2Mpolynucleotides of the invention. For example, such host cells cancontain variant A2M polynucleotides introduced into the host cell usingknown transformation, transfection or infection methods. As used herein,a cell capable of expressing a variant A2M polynucleotide can be“transformed.” The variant A2M polypeptides of the invention can beprepared by culturing transformed host cells under culture conditionssuitable to express the recombinant protein. Any procedure forintroducing foreign nucleotide sequences into host cells may be used.Non-limiting examples include the use of calcium phosphate transfection,transfection, DEAE, dextran-mediated transfection, microinjection,lipofection, polybrene, protoplast fusion, electroporation (Davis, L. etal., Basic Methods in Molecular Biology (1986)), liposomes,microinjection, plasma vectors, viral vectors, and any other well-knownmethods for introducing cloned genomic DNA, cDNA, synthetic DNA, orother foreign genetic material into a host cell. A genetic engineeringprocedure capable of successfully introducing at least one gene into thehost cell capable of expressing the variant A2M polynucleotide can beused.

The present invention still further provides host cells engineered toexpress the variant A2M polynucleotides of the invention, wherein thevariant A2M polynucleotides are operative with a regulatory sequenceheterologous to the host cell which drives expression of the variant A2Mpolynucleotides in the cell. Knowledge of A2M-like DNA allows formodification of cells to permit, or increase, expression of A2M-likepolypeptide. Cells can be modified, for example, by homologousrecombination, to provide increased variant A2M polypeptide expressionby replacing, in whole or in part, the naturally occurring A2M derivedfrom the SV40 viral genome, for example, SV40 macroglobulin-likepromoter with all or part of a heterologous promoter so that the cells'variant A2M sites can be used to provide the required non-transcribedpolypeptide and can be expressed at higher levels.

For long-term, high-yield production of recombinant variant A2Mpolypeptides, stable expression is preferred. For example, cell linesthat stably express the variant A2M sequences described herein can beengineered. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA, engineered cells are allowed to grow for 1-2 days in anenriched media, and then are switched to a selective media. Theselectable marker in the recombinant plasmid confers resistance to theselection and allows cells to stably integrate the plasmid into theirchromosomes and grow to form foci which in turn are cloned and expandedinto cell lines. This method is advantageously used to engineer celllines which express the variant A2M gene product. Such engineered celllines are particularly useful in screening and evaluation of compoundsthat affect the endogenous activity of the variant A2M gene product. Anumber of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase, hypoxanthine-guaninephosphoribosyltransferase, and adenine phosphoribosyltransferase genescan be employed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate; gpt,which confers resistance to mycophenolic acid; neo, which confersresistance to the aminoglycoside G-418; and hygro, which confersresistance to hygromycin.

Variant A2M polynucleotide sequences can be engineered so as to modifyprocessing or expression of the protein. For example, and not by way oflimitation, the variant A2M polynucleotides can be combined with apromoter sequence and/or ribosome binding site, or a signal sequence canbe inserted upstream of variant A2M polynucleotide sequences to permitsecretion of the variant A2M polypeptide and thereby facilitateharvesting or bioavailability. Additionally, a variant A2Mpolynucleotide can be mutated in vitro or in vivo, to create and/ordestroy translation, initiation, and/or termination sequences, or tocreate variations in coding regions and/or form new restriction sites ordestroy preexisting ones, or to facilitate further in vitromodification. Any technique for mutagenesis known in the art can beused, including but not limited to, in vitro site-directed mutagenesis.

Further, nucleic acids encoding other proteins or domains of otherproteins can be joined to nucleic acids encoding variant A2Mpolypeptides or fragments thereof so as to create a fusion protein.Nucleotides encoding fusion proteins can include, but are not limitedto, a full length variant or wild-type A2M protein, a truncated variantor wild-type A2M protein or a peptide fragment of a variant or wild typeA2M protein fused to an unrelated protein or peptide, such as forexample, a transmembrane sequence, which anchors the A2M peptidefragment to the cell membrane; an Ig Fc domain which increases thestability and half-life of the resulting fusion protein; maltose bindingprotein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX), aHis tag, an enzyme, fluorescent protein, luminescent protein which canbe used as a marker, for example, an A2M-Green Fluorescent Proteinfusion protein. The fusion proteins can be used for affinitypurification.

The variant A2M nucleic acids and polypeptides can also be expressed inorganisms so as to create a transgenic organism. Desirable transgenicplant systems having one or more of these sequences include Arabidopsis,Maize, and Chlamydomonas. Desirable insect systems having one or more ofthe variant A2M polynucleotides and/or polypeptides include, forexample, D. melanogaster and C. elegans. Animals of any species,including, but not limited to, amphibians, reptiles, birds, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, dogs, cats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees can be used togenerate variant A2M containing transgenic animals. Transgenic organismsdesirably exhibit germline transfer of variant A2M nucleic acids andpolypeptides described herein.

A variety of methodologies known in the art can be utilized to obtainany one of the isolated polypeptides or proteins of the presentinvention. At the simplest level, the amino acid sequence can besynthesized using commercially available peptide synthesizers. Thesynthetically constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins can possess biological properties incommon therewith, including protein activity. This technique can beparticularly useful in producing small peptides and fragments of largerpolypeptides. Fragments are useful, for example, in generatingantibodies against the native polypeptide. Thus, they can be employed asbiologically active or immunological substitutes for natural, purifiedproteins in screening of therapeutic compounds and in immunologicalprocesses for the development of antibodies. The polypeptides andproteins of the present invention can alternatively be purified fromcells which have been altered to express the desired polypeptide orprotein. As used herein, a cell can be said to be altered to express adesired polypeptide or protein when the cell, through geneticmanipulation, can be made to produce a polypeptide or protein which itnormally does not produce or which the cell normally produces at a lowerlevel. One skilled in the art can readily adapt procedures forintroducing and expressing either recombinant or synthetic sequencesinto eukaryotic or prokaryotic cells in order to generate a cell whichproduces one of the polypeptides or proteins of the present invention.

The invention also relates to methods for producing a polypeptidecomprising growing a culture of host cells in a suitable culture medium,and purifying the protein from the cells or the culture in which thecells are grown. For example, the methods can include a process forproducing a polypeptide in which a host cell containing a suitableexpression vector that includes a polynucleotide of the invention can becultured under conditions that allow expression of the encodedpolypeptide. The polypeptide can be recovered from the culture,conveniently from the culture medium, or from a lysate prepared from thehost cells and further purified. Preferred embodiments include those inwhich the protein produced by such process can be a full length ormature form of the protein, such as A2M. In an alternative method, thepolypeptide or protein can be purified from bacterial cells whichnaturally produce the polypeptide or protein. One skilled in the art canreadily follow known methods for isolating polypeptides and proteins inorder to obtain one of the isolated polypeptides or proteins of thepresent invention. These include, but are not limited to,immunochromatography, HPLC, size-exclusion chromatography, ion-exchangechromatography, and immuno-affinity chromatography. See, e.g., Scopes,Protein Purification: Principles and Practice, Springer-Verlag (1994);Sambrook, et al., in Molecular Cloning: A Laboratory Manual; Ausubel etal., Current Protocols in Molecular Biology. Polypeptide fragments thatretain biological or immunological activity include fragments comprisinggreater than about 100 amino acids, or greater than about 200 aminoacids, and fragments that encode specific protein domains. The purifiedpolypeptides can be used in in vitro binding assays which are well knownin the art to identify molecules which bind to the polypeptides. Thesemolecules include, but are not limited to, small molecules, moleculesfrom combinatorial libraries, antibodies or other proteins. Themolecules identified in a binding assay can then be tested forantagonist or agonist activity in in vivo tissue culture or animalmodels that are well known in the art. In brief, the molecules can betitrated into a plurality of cell cultures or animals and then testedfor either cell or animal death or prolonged survival of the animal orcells.

The resulting expressed variant A2M polypeptides can then be purifiedfrom a culture, for example, from culture medium or cell extracts, usingknown purification processes, such as affinity chromatography, gelfiltration, and ion exchange chromatography. The purification of thevariant A2M polypeptides can also include an affinity column containingagents which will bind to the protein; one or more column steps oversuch affinity resins as concanavalin A-agarose, heparin-Toyopearl™ orCibacron blue 3GA Sepharose™; one or more steps involving hydrophobicinteraction chromatography using such resins as phenyl ether, butylether, or propyl ether; or immunoaffinity chromatography. Alternatively,the protein of the invention can also be expressed in a form which willfacilitate purification. For example, a protein can be expressed as afusion protein, such as those of maltose binding protein (MBP),glutathione-S-transferase (GST) or thioredoxin (TRX), or as a His tag.Kits for expression and purification of such fusion proteins arecommercially available from New England BioLab (Beverly, Mass.),Pharmacia (Piscataway, N.J.) and Invitrogen, respectively. The proteincan also be tagged with an epitope and subsequently purified by using aspecific antibody directed to such epitope. One such epitope (“FLAG®”)is commercially available from Kodak (New Haven, Conn.). Finally, one ormore reverse-phase high performance liquid chromatography (RP-HPLC)steps employing hydrophobic RP-HPLC media, for example, silica gelhaving pendant methyl or other aliphatic groups, can be employed tofurther purify the protein. Any combination of the foregoingpurification procedures can also be employed to provide a substantiallyhomogeneous isolated or purified recombinant variant A2M polypeptide.The variant A2M polypeptides purified can be substantially free of othermammalian proteins and can be defined in accordance with the presentinvention as an “isolated protein.”

Therapeutic Methods

Any method known in the art can be used to treat the chronic wound, orto treat the pathology that can be causing the chronic wound. A methodcan comprise treatment of a chronic wound in a mammal, such as aneuropathic ulcer decubitus ulcer, a venous ulcer or a diabetic ulcer oran infected wound. The method can comprise applying an A2M compositionto the wound. A2M compositions and formulations can be used forinhibiting proteases. A2M compositions can be used to prevent, slow, oralter FAC formation. A variant A2M can be more efficient than awild-type A2M polypeptide in inhibiting proteases, have a longerhalf-life, have a slower clearance factor, or any combination thereof.

In some embodiments, the wound is a decubital ulcer, a pressure ulcer, alower extremity ulcer, a deep sternal wound, a post-operative wound, arefractory post-operative wound of the trunk area, a wound to the greatsaphenous vein following harvesting of the great saphenous vein, avenous ulcer, or an anal fissure. In those embodiments involving a lowerextremity ulcer, the ulcer may be in a diabetic patient. In otherembodiments, the wound is a venous ulcer, pressure ulcer, orpost-operative ulcer.

The A2M composition can be comprised on a wound dressing. Dry andhydrated, i.e. wet wound dressings and delivery systems can be used andcan also be suitable for active ingredients, their use for the treatmentof wounds and skin diseases, preferably chronic wounds. A wound dressingcan be applied to the chronic wound for a period of at least 1 hour, atleast 24 hours, at least 48 hours, or at least 72 hours. The treatmentmay be extended for several days, weeks or months, with dressing changesas appropriate, if necessary for chronic wounds.

Another aspect of the invention relates to articles of manufacturecomprising a composition of the invention and a dressing. In someembodiments, the dressing is a dry dressing, moisture-keeping barrierdressing, or bioactive dressing. In those embodiments involving a drydressing, the dressing may be gauze, a bandage, a non-adhesive mesh, amembrane, foils, foam, or a tissue adhesive. In those embodimentsinvolving a moisture-keeping barrier dressing, the dressing may be apaste, a cream, an ointment, a nonpermeable or semi-permeable membraneor foil, a hydrocolloid, a hydrogel, or combinations thereof. In thoseembodiments involving a bioactive dressing, the dressing may be anantimicrobial dressing. For example, the wound dressing may be a woven,nonwoven or knitted fabric having the A2M composition coated thereon, orit may be a bioresorbable polymer film or sponge having the A2Mcomposition dispersed therein for sustained release at the ulcer site.

Once the site from which the pain can be originating can be identifiedby the presence of A2M, any method known in the art can be used to treatthe pain, or to treat the pathology that can be causing the pain. Forexample, if radiculopathy or discogenic pain or facet pain has beendiagnosed, any number of methods known in the art for treating spinalpain can be applied to treat the patient. Suitable methods include, butare not limited to, laminotomy, laminectomy, discectomy,microdiscectomy, percutaneous discectomy, endoscopic discectomy, laserdiscectomy, foramenotomy, fusion, prolotherapy, other surgicaldecompressions, decompression with fusion with or withoutinstrumentation.

Pain in the spine can also be treated by standard non-surgical methods,including administration of steroidal or non-steroidal anti-inflammatoryagents. Non-steroidal anti-inflammatory (NSAID) agents are well known inthe art. Non-steroidal agents, including NSAIDs such as ibuprofen,aspirin or paracetamol can be used. Steroids, such as glucocorticoids,which reduce inflammation by binding to cortisol receptors, can also beused for treatment.

Any number of methods known in the art for treating joint-related paincan be applied to treat the patient. Suitable methods include surgicaland non-surgical methods including, but not limited to, arthroscopicdebridement or administration of steroidal or non-steroidalanti-inflammatory agents.

Any of the compositions described herein can be used for enhancing thenonspecific inhibition of one or more proteases in a human or non-humananimal experiencing or susceptible to one or more conditions selectedfrom the group of arthritis, inflammation, ligament injury, tendoninjury, bone injury, cartilage degeneration, cartilage injury, anautoimmune disease, back pain, joint pain, joint degeneration, discdegeneration, spine degeneration, bone degeneration, or any combinationthereof. A variant A2M polypeptide can be administered to an animal toreduce one or more protease activities in an animal.

A variant A2M polypeptide can be used for inhibiting proteases. Avariant A2M polypeptide can be used for treatment of pain andinflammation conditions and diseases. A variant A2M polypeptide can beused to prevent, slow, or alter FAC formation. A variant A2M can be moreefficient than a wild-type A2M polypeptide in inhibiting proteases, havea longer half-life, have a slower clearance factor, or any combinationthereof.

A variant A2M polypeptide can be for administration by parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), transdermal (either passively or using iontophoresis orelectroporation), or transmucosal (nasal, vaginal, rectal, orsublingual), oral, intra-articular or inhalation routes ofadministration. A variant A2M polypeptide can also be administered usingbioerodible inserts, bare-metal stents (BMS), or drug-eluting stents(DES or coated stents, or medicated stents), and can be delivereddirectly to spinal structures, such as intervertebral discs, theepidural space and facet joints, or to diarthroidal joints. A variantA2M polypeptide can be formulated in dosage forms appropriate for eachroute of administration. A variant A2M polypeptide can additionally beformulated for enteral administration.

A variant A2M polypeptide can be administered to a subject in atherapeutically effective amount. The precise dosage will vary accordingto a variety of factors such as subject dependent variables, such asage, the injury or pathology being treated, and the treatment beingaffected. The exact dosage can be chosen by the individual physician inview of the patient to be treated. Dosage and administration areadjusted to provide sufficient levels of the active moiety or tomaintain the desired effect. Additional factors that can be taken intoaccount include the severity of the disease, age of the organism, andweight or size of the organism; diet, time and frequency ofadministration, drug combination(s), reaction sensitivities, andtolerance/response to therapy. Short acting pharmaceutical compositionsare administered daily whereas long acting pharmaceutical compositionsare administered every 2, 3 to 4 days, every week, or once every twoweeks. Depending on half-life and clearance rate of the particularformulation, the pharmaceutical compositions of the invention areadministered once, twice, three, four, five, six, seven, eight, nine,ten or more times per day.

For some compositions, such a variant A2M polypeptide disclosed herein,as further studies are conducted information will emerge regardingappropriate dosage levels for treatment of various conditions in varioussubjects, and the ordinary skilled worker, considering the therapeuticcontext, age, and general health of the recipient, will be able toascertain proper dosing. The selected dosage depends upon the route ofadministration, and on the duration of the treatment desired. Generallydosage levels can include 0.1 to 40 mg/kg of body weight daily.Generally, for local injection or infusion, dosages can be lower.Depending on the composition and site of administration, dosage levelscan be between about 1 to 500,000 mg, in a volume between about 0.1 to10 mL. For example, dosage levels can be between about 5 to 450 mg, 5 to400 mg, 5 to 350 mg, 5 to 300 mg, 5 to 250 mg, 5 to 200 mg, 5 to 150 mg,5 to 100 mg, 5 to 500 mg, 5 to 25 mg, 100 to 150 mg, 100 to 200 mg, 100to 250 mg, 100 to 300 mg, 100 to 350 mg, 100 to 400 mg, 100 to 450 mg,or 100 to 500 mg in a volume between about 0.1 to 9 mL, 0.1 to 8 mL, 0.1to 7 mL, 0.1 to 6 mL, 0.1 to 5 mL, 0.1 to 4 mL, 0.1 to 3 mL, 0.1 to 2mL, 0.1 to 1 mL, 0.1 to 0.9 mL, 0.1 to 0.7 mL, 0.1 to 0.6 mL, 0.1 to 0.5mL, 0.1 to 0.4 mL, 0.1 to 0.3 mL, 0.1 to 0.2 mL, 1 to 9 mL, 1 to 8 mL, 1to 7 mL, 1 to 6 mL, 1 to 5 mL, 1 to 4 mL, 1 to 3 mL, or 1 to 2 mL.Normal dosage amounts of various variant A2M polypeptides or nucleicacids, or fragment thereof can vary from any number betweenapproximately 1 to 500,000 micrograms, up to a total dose of about 50grams, depending upon the route of administration. Desirable dosagesinclude, for example, 250 μg, 500 μg, 1 mg, 50 mg, 100 mg, 150 mg, 200mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 1 g, 1.1 g, 1.2 g, 1.3 g,1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2 g, 3 g, 4 g, 5, 6 g, 7 g, 8g, 9 g, 10 g, 20 g, 30 g, 40 g, and 50 g.

The dose of the variant A2M polypeptide, or fragment thereof, can beadministered to produce a tissue or blood concentration or both fromapproximately any number between 0.1 μM to 500 mM. Desirable dosesproduce a tissue or blood concentration or both of about any numberbetween 1 to 800 μM. Preferable doses produce a tissue or bloodconcentration of greater than about any number between 10 μM to about500 μm. Preferable doses are, for example, the amount of activeingredient required to achieve a tissue or blood concentration, or both,of 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM,60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 110 μM,120 μM, 130 μM, 140 μM, 145 μM, 150 μM, 160 μM, 170 μM, 180 μM, 190 μM,200 μM, 220 μM, 240 μM, 250 μM, 260 μM, 280 μM, 300 μM, 320 μM, 340 μM,360 μM, 380 μM, 400 μM, 420 μM, 440 μM, 460 μM, 480 μM, and 500 μM.Although doses that produce a tissue concentration of greater than 800μM are not preferred, they can be used with some embodiments of theinvention. A constant infusion of the variant A2M polypeptide, orfragment thereof, can also be provided so as to maintain a stableconcentration in the tissues as measured by blood levels.

A variant A2M polypeptide can be administered in an aqueous solution byparenteral, intradiscal, intrafacet, intrathecal, epidural or jointinjection. A variant A2M polypeptide herein can be administered directlyinto the area of the spine or joint that can be the source of pain inthe subject. For example, when fibronectin-aggrecan complexes aredetected in the epidural space, a variant A2M polypeptide that inhibitsproteases or that prevents FAC formation can be administered by directinjection into the epidural space. Alternatively, variant A2Mpolypeptide that inhibits proteases or that prevents FAC formation canbe administered by direct injection into the disc space, facet joint, ordiarthroidial joint when fibronectin-aggrecan complexes are detected inthese spaces. In some embodiments, aggrecan can include anynaturally-occurring variants and splice variants of aggrecan, versican,brevican and neurocan, and any variants of aggrecan, versican, brevicanand neurocan due to splicing by different cell types. In someembodiments, fibronectin can include any naturally occurring fibronectinvariants including approximately 20 known splice variants associatedwith a disease or a disorder and fibronectin variants due to differentsplicing by different cell types.

A composition or formulation or agent can also be in the form of asuspension or emulsion. In general, pharmaceutical compositions areprovided including effective amounts of a peptide or polypeptide, andoptionally include pharmaceutically acceptable diluents, preservatives,solubilizers, emulsifiers, adjuvants and/or carriers. Such compositionsinclude diluents sterile water, buffered saline of various buffercontent (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; andoptionally, additives such as detergents and solubilizing agents (e.g.,TWEEN® 20, TWEEN® 80, Polysorbate 80), anti-oxidants (e.g., ascorbicacid, sodium metabisulfite), and preservatives (e.g., Thimersol, benzylalcohol) and bulking substances (e.g., lactose, mannitol). Examples ofnon-aqueous solvents or vehicles are propylene glycol, polyethyleneglycol, vegetable oils, such as olive oil and corn oil, gelatin, andinjectable organic esters such as ethyl oleate. The formulations can belyophilized and redissolved or resuspended immediately before use. Theformulation can be sterilized by, for example, filtration through abacteria retaining filter, by incorporating sterilizing agents into thecompositions, by irradiating the compositions, or by heating thecompositions.

A variant A2M polypeptide can also be administered in controlled releaseformulations. Controlled release polymeric devices can be made for longterm release systemically following implantation of a polymeric device(rod, cylinder, film, or disc) or injection (microparticles). The matrixcan be in the form of microparticles such as microspheres, wherepeptides are dispersed within a solid polymeric matrix or microcapsules,where the core can be of a different material than the polymeric shell,and the peptide can be dispersed or suspended in the core, which can beliquid or solid in nature. Unless specifically defined herein,microparticles, microspheres, and microcapsules are usedinterchangeably. Alternatively, the polymer can be cast as a thin slabor film, ranging from nanometers to four centimeters, a powder producedby grinding or other standard techniques, or even a gel such as ahydrogel.

Either non-biodegradable or biodegradable matrices can be used fordelivery of any composition described herein, although biodegradablematrices are preferred. These can be natural or synthetic polymers,although synthetic polymers are preferred due to the bettercharacterization of degradation and release profiles. The polymer can beselected based on the period over which release can be desired. In somecases linear release can be most useful, although in others a pulserelease or “bulk release” can provide more effective results. Thepolymer can be in the form of a hydro gel (typically in absorbing up toabout 90% by weight of water), and can optionally be crosslinked withmultivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release, 5:13-22(1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

The devices can be formulated for local release to treat the area ofimplantation or injection which will typically deliver a dosage that canbe much less than the dosage for treatment of an entire body or systemicdelivery. These can be implanted or injected subcutaneously, into themuscle, fat, or swallowed.

A variant A2M polypeptide can be used in the treatment of a condition ora disease, such as a chronic wound. For example, a condition or diseasecan be tendon condition, ligament condition, joint injury, spine injury,or inflammation, Alzheimer's disease, cerebral amyloid angiopathy,multiple sclerosis, congenital anti-thrombin deficiency, rheumatoidarthritis, growth of various tumors, coronary or limb ischemia,retinopathies, and regulation of immune response to tumors and viralinfections. Others include Acne vulgaris, Alzheimer's disease,arthritis, asthma, acne, allergies and sensitivities, Autoimmunediseases, atherosclerosis, bronchitis, cancer, carditis, Crohn'sdisease, colitis, chronic pain, cirrhosis, Celiac disease, Chronicprostatitis, dermatitis diverticulitis, dementia, dermatitis, diabetes,dry eyes, edema, emphysema, eczema, fibromyalgia, gastroenteritis,gingivitis, Glomerulonephritis, Hypersensitivities, hepatitislupuserythematous, acid reflux/heartburn, heart disease, hepatitis, highblood pressure, insulin resistance, Interstitial cystitis, Inflammatorybowel diseases, irritable bowel syndrome (IBS), jointpain/arthritis/rheumatoid arthritis, metabolic syndrome (syndrome X),myositis, nephritis, obesity, osteopenia, osteoporosis, Pelvicinflammatory disease, Parkinson's disease, periodontal disease,polyarteritis, polychondritis, psoriasis, Reperfusion injury, Rheumatoidarthritis, Sarcoidosis, scleroderma, sinusitis, Sjögren's syndrome,spastic colon, systemic candidiasis, tendonitis, Transplant rejection,ulcerative colitcis, UTI's, Vasculitis, and vaginitis.

In some embodiments, a variant A2M polypeptide, can be used in thetreatment of cancer. For example, variant A2M polypeptides can beadministered directly into a tumor, such as a solid tumor, by injectionor another suitable means.

An autoimmune disease can be a disease or disorder arising from anddirected against an individual's own tissues or organs or a co-segregateor manifestation thereof or resulting condition therefrom. In many ofthese autoimmune and inflammatory disorders, a number of clinical andlaboratory markers can exist, including, but not limited to,hypergammaglobulinemia, high levels of auto-antibodies, antigen-antibodycomplex deposits in tissues, benefit from corticosteroid orimmunosuppressive treatments, and lymphoid cell aggregates in affectedtissues. Without being limited to any one theory regarding B-cellmediated autoimmune disease, it is believed that B-cells demonstrate apathogenic effect in human autoimmune diseases through a multitude ofmechanistic pathways, including autoantibody production, immune complexformation, dendritic and T-cell activation, cytokine synthesis, directchemokine release, and providing a nidus for ectopic neo-lymphogenesis.

Each of these pathways can participate to different degrees in thepathology of autoimmune diseases. “Autoimmune disease” can be anorgan-specific disease (i.e., the immune response can be specificallydirected against an organ system such as the endocrine system, thehematopoietic system, the skin, the cardiopulmonary system, thegastrointestinal and liver systems, the renal system, the thyroid, theears, the neuromuscular system, the central nervous system, etc.) or asystemic disease that can affect multiple organ systems (for example,SLE, RA, polymyositis, etc.). Preferred such diseases include autoimmunerheumatologic disorders (such as, for example, RA, Sjogren's syndrome,scleroderma, lupus such as SLE and lupus nephritis, polymyositis,dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome,and psoriatic arthritis), autoimmune gastrointestinal and liverdisorders (such as, for example, inflammatory bowel diseases (e.g.,ulcerative colitis and Crohn's disease), autoimmune gastritis andpernicious anemia, autoimmune hepatitis, primary biliary cirrhosis,primary sclerosing cholangitis, and celiac disease), vasculitis be (suchas, for example, ANCA-negative vasculitis and ANCA-associatedvasculitis, including Churg-Strauss vasculitis, Wegener'sgranulomatosis, and microscopic polyangiitis), autoimmune neurologicaldisorders (such as, for example, MS, opsoclonus myoclonus syndrome,myasthenia gravis, neuromyelitis optica, Parkinson's disease,Alzheimer's disease, and autoimmune polyneuropathies), renal disorders(such as, for example, glomerulonephritis, Goodpasture's syndrome, andBerger's disease), autoimmune dermatologic disorders (such as, forexample, psoriasis, urticaria, hives, pemphigus vulgaris, bullouspemphigoid, and cutaneous lupus erythematosus), hematologic disorders(such as, for example, thrombocytopenic purpura, thromboticthrombocytopenic purpura, posttransfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases(such as, for example, inner ear disease and hearing loss), Behcet'sdisease, Raynaud's syndrome, organ transplant, and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as insulin-dependent diabetes mellitus (IDDM), Addison's disease,and autoimmune thyroid disease (e.g., Graves' disease and thyroiditis)).More preferred such diseases include, for example, RA, ulcerativecolitis, ANCA-associated vasculitis, lupus, MS, Sjogren's syndrome,Graves' disease, IDDM, pernicious anemia, thyroiditis, andglomerulonephritis.

A variant A2M polypeptide can be suitable for delivery into one or morejoints or into the spine. One or more joints can be one or moresynovial, diarthrodial, amphiarthrodial, synarthrodial, symphyseal, orcartilaginous joints. A joint can be a wrist, spinal, vertebral,cervical, shoulder, elbow, carpal, metacarpal, phalangeal,acromioclavicular, sternoclavicular, scapular, costal, sacroiliac, hip,knee, ankle tarsal, articulations of a foot or hand, axillaryarticulations, or a metatarsal.

A joint can refer to any diarthoidal (also called synovial) joints. Ajoint can be any joint containing bone, articular cartilage, a jointcapsule, a synovial tissue lining, or lubricating synovial fluid insidea joint capsule. Cartilage components of a joint can be a chondralcomponent. A component of the knee can be a meniscal component. In someembodiments, a synovial joint can be a shoulder or wrist or ankle or hipor elbow, or the small joints of the fingers or toes. A joint can be anormal joint or a control joint. A normal or control joint can be ajoint that can be an insignificant source of pain to a subject. Thelevel of pain that can be present in a normal joint typically may notimpact the function or quality of the patient's life to the degree thatthe patient seeks medical care. A joint sample or sample from a jointcan be a sample of tissue or fluid from a joint including, but notlimited to, ex vivo and in vivo synovial fluid samples and joint ortissue lavages. A joint sample or sample from a joint can be abiological sample.

A variant A2M polypeptide can be used in the treatment of pain, such aspain associated with a condition or a disease of the current disclosure.

Pain can be radicular pain, radiculopathy, radiculopathic pain andsciatica and can be radiating pain of the extremities which emanatesfrom the spinal root level or “radic” along the path of one or moreirritated lumbar nerve roots. In the case of sciatica, this canoriginate from the L4, L5 and/or L6 or transitional vertebrae if presentand/or sacroiliac spinal nerve roots, which make up the sciatic nerve.Radiating pain can be also possible from the high lumbar discherniations in the 13, 12 or 11 regions or from any cervical nerve rootin the case of a cervical disc herniation, cervical nerve rootirritation or cervical disc degeneration. This pain can differ from painresulting from a facet joint or other spinal structure, which can beclassified as “referred” pain. Radiating pain can be also possible fromthe high lumbar disc herniations in the L3, L2 or L1 regions or cervicalspine regions.

Pain can be discogenic pain and can be spinal related pain thatgenerates from an intervertebral disc. The intervertebral disc suffersfrom reduced functionality in association with a loss of hydration fromthe nucleus pulposus. The reduction in functionality coincides withdamage in the annulus fibrosus. This weakening can lead to anatomiclesions such as bulging, prolapsed, extruded, or sequestered disc. Thisweakening can also lead to possible biochemical lesions resulting fromleakage of the disc contents that can manifest in back pain oraforementioned chemical radiculopathy.

Pain can be facet joint pain or facetogenic pain and can be paingenerating from a facet joint, facet joints, or zygapophysial jointsthat are paired, true synovial joints endowed with cartilage, capsule,meniscoid, and synovial membrane. Spinal-pain or spine related painincludes, but is not limited to, discogenic, facetogenic andradiculopathic pain.

Pain can be acute pain and can be pain lasting up to six months, e.g.,five months, four months, three months, two months, four weeks, threeweeks, two weeks, one week, six days, five days, four days, three days,two days or one day or less. Chronic pain can be pain of duration longerthan six months.

Any subject described herein can be treated with any of the compositionsdescribed herein. In some embodiments, a subject can be diagnosed with acondition or disease before or after being diagnosed with a condition ordisease, such as by the methods described in U.S. Pat. No. 7,709,215 andU.S. Publication No.: US 2010/0098684A1. In some embodiments, a subjectcan be treated with any composition described herein, before or afterbeing diagnosed with a condition or disease.

Subjects

Subjects can include any subject that presents with pain in the spine orjoint. In some embodiments, a subject can be selected for the detectionof A2M. Preferably the subject can be human. Subjects can beexperiencing any pain, such as pain associated with the spine,including, but not limited to, discogenic, facetogenic or radiculopathicpain.

Subjects can be suspected of experiencing pain associated with anyanatomic structure of a joint including, but not limited to, bone,articular cartilage, or the synovial tissue lining. Joints can include,but are not limited to, large diarthrodial (synovial) joints (e.g. knee,hip, shoulder), small diarthrodial (synovial) joints (e.g. elbow, wrist,ankle, zygoapophyseal or facet joints of spine), and amphiarthrodialjoints (e.g. sacroiliac joint, sternoclavicular joint, tempomandibularjoint (“TMJ”)). Subjects can be experiencing acute joint-related pain,or can suffer from chronic joint-related pain. These can be related todegenerative disease (e.g. osteoarthritis), myofascial pain syndromes,inflammatory or crystalline arthritides, or other enthesopathies,tendon/ligament injuries or degeneration, or soft tissue pathologyoutside the musculoskeletal system.

In some embodiments, a subject may have been experiencing joint-relatedor spine-related pain for 30 or 25 weeks or less. In some embodiments, asubject may have been experiencing joint-related or spine-related painfor 20, 15, 10, 8, or 6 weeks, or less. Subjects can be of either sexand can be of any age. Subjects may be experiencing acute or chronicpain.

A subject can be human or non-human animal. For example, the animal canbe a mammal, such as a mouse, rat, rabbit, cat, dog, monkey, horse orgoat. A subject can be a virus, bacterium, mycoplasma, parasite, fungus,or plant, or animal, such as a mammal, for example, a human.

In some embodiments, a subject can be diagnosed as needing treatmentwith any of the compositions described herein. For example, a subjectcan be diagnosed as needing treatment with an A2M enriched sample or anagent that can prevent FAC formation.

Samples

Any of the compositions described herein can be derived from abiological sample. Biological samples can also include sections oftissues such as biopsy samples, frozen sections taken for histologicpurposes, and lavage samples. A biological sample can be from a virus,bacterium, mycoplasma, parasite, fungus, or plant. A biological samplecan be from an animal, such as a mammal, for example, a human, non-humanprimate, rodent, caprine, bovine, ovine, equine, canine, feline, mouse,rat, rabbit, horse or goat.

A biological sample can be a tissue sample or bodily fluid, such as ahuman bodily fluid. For example, the bodily fluid can be blood, sera,plasma, lavage, urine, cerebrospinal fluid (CSF), sputum, saliva, bonemarrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breastmilk, broncheoalveolar lavage fluid, semen, prostatic fluid, Cowper'sfluid, pre-ejaculatory fluid, female ejaculate, sweat, tears, cystfluid, pleural fluid, peritoneal fluid, pericardial fluid, lymph, chyme,chyle, bile, interstitial fluid, menses, pus, sebum, vaginal secretion,mucosal secretion, stool water, pancreatic juice, lavage fluid fromsinus cavities, bronchopulmonary aspirate, blastocyl cavity fluid, orumbilical cord blood. One or more of the biological sample(s) cancomprise a cell, such as a stem cell, undifferentiated cell,differentiated cell, or cell from a diseased subject or subject with aspecific condition. A biological sample can be blood, a cell, apopulation of cells, a quantity of tissue, fluid, or lavasate from ajoint of a subject. A biological sample can comprise cells fromcartilaginous tissue or can be free of cells. A biological sample can besubstantially depleted of a common serum protein, such as, but notlimited to, albumin or IgG. Depletion can comprise filtration,fractionation, or affinity purification.

Biological samples can be collected by any non-invasive means, such as,for example, by drawing blood from a subject, or using fine needleaspiration or needle biopsy. Alternatively, biological samples can becollected by an invasive method, including, for example, surgicalbiopsy.

A biological sample can comprise disease or condition specific proteins.A biological sample can be from a subject with a disease or condition orfrom a subject without a disease or condition. In some embodiments, abiological sample can be from a subject diagnosed with a disease orcondition or from a subject not diagnosed with or without a disease orcondition. A diagnosis can be made by any of the methods describedherein. A biological sample can be from a subject at one time point andanother biological sample can be from a subject at a later or earliertime point, wherein the subject can be the same or a different subject.For example, the subject may have a disease or condition or have beendiagnosed with a disease or condition, and samples can be taken as thedisease or condition progresses. A biological sample can be from asubject pretreatment and another biological sample can be from a subjectat post treatment, wherein the subject can be the same or differentsubject. A biological sample can be from a subject non-responsive totreatment and another biological sample can be from a subject responsiveto a treatment. Biological samples can be from the same or differentspecies. One or more biological samples can be from the same subject orfrom a different subject from which one or more other biological sampleswere obtained.

A spine sample or sample from the spine can be a sample of tissue orfluid from the spine or added to the spine (lavage) including, but notlimited to, spinal disc samples, epidural samples, and facet jointsamples. A spine sample or sample from the spine can be a biologicalsample. Any number of methods known in the art can be used to retrievesample from the spine for the detection of inflammation biomarkers.These methods include, but are not limited to, methods for obtainingsamples from the epidural space, the intervertebral disc space and thefacet joint space. Any number of methods known in the art can be used toobtain joint samples for the detection of inflammation biomarkers.Suitable methods include, but are not limited to, percutaneous or openaspiration, biopsy, or lavage.

The methods of the invention can be applied to the study of any type ofbiological samples allowing one or more biomarkers to be assayed. Abiological sample can be a fresh or frozen sample collected from asubject, or archival samples with known diagnosis, treatment and/oroutcome history.

The inventive methods can be performed on the biological sample itselfwithout or with limited processing of the sample. The inventive methodscan be performed at the single cell level (e.g., isolation of cells fromthe biological sample). Multiple biological samples can be taken fromthe same tissue/body part in order to obtain a representative samplingof the tissue.

Any of the method described herein can be performed on a protein extractprepared from the biological sample. The methods can also be performedon extracts containing one or more of: membrane proteins, nuclearproteins, and cytosolic proteins. Methods of protein extraction are wellknown in the art (see, for example “Protein Methods”, D. M. Bollag etal., 2nd Ed., 1996, Wiley-Liss; “Protein Purification Methods: APractical Approach”, E. L. Harris and S. Angal (Eds.), 1989; “ProteinPurification Techniques: A Practical Approach”, S. Roe, 2nd Ed., 2001,Oxford University Press; “Principles and Reactions o/Protein Extraction,Purification, and Characterization”, H. Ahmed, 2005, CRC Press: BocaRaton, Fla.). Numerous different and versatile kits can be used toextract proteins from bodily fluids and tissues, and are commerciallyavailable from, for example, BioRad Laboratories (Hercules, Calif.), BDBiosciences Clontech (Mountain View, Calif.), Chemicon International,Inc. (Temecula, Calif.), Calbiochem (San Diego, Calif.), PierceBiotechnology (Rockford, Ill.), and Invitrogen Corp. (Carlsbad, Calif.).After the protein extract has been obtained, the protein concentrationof the extract can be standardized to a value being the same as that ofthe control sample in order to allow signals of the protein markers tobe quantitated. Such standardization can be made using photometric orspectrometric methods or gel electrophoresis.

Any of the method described herein can be performed on nucleic acidmolecules extracted from the biological sample. For example, RNA can beextracted from the sample before analysis. Methods of RNA extraction arewell known in the art (see, for example, J. Sambrook et al., “MolecularCloning: A Laboratory Manual”, 1989, 2nd Ed., Cold Spring HarborLaboratory Press: Cold Spring Harbor, N.Y.). Most methods of RNAisolation from bodily fluids or tissues are based on the disruption ofthe tissue in the presence of protein denaturants to quickly andeffectively inactivate RNAses. Isolated total RNA can then be furtherpurified from the protein contaminants and concentrated by selectiveethanol precipitations, phenol/chloroform extractions followed byisopropanol precipitation or cesium chloride, lithium chloride or cesiumtrifluoroacetate gradient centrifugations. Kits are also available toextract RNA (i.e., total RNA or mRNA) from bodily fluids or tissues andare commercially available from, for example, Ambion, Inc. (Austin,Tex.), Amersham Biosciences (Piscataway, N.J.), BD Biosciences Clontech(Palo Alto, Calif.), BioRad Laboratories (Hercules, Calif.), GIBCO BRL(Gaithersburg, Md.), and Qiagen, Inc. (Valencia, Calif.).

After extraction, mRNA can be amplified, and transcribed into cDNA,which can then serve as template for multiple rounds of transcription bythe appropriate RNA polymerase. Amplification methods are well known inthe art (see, for example, A. R. Kimmel and S. L. Berger, MethodsEnzymol. 1987, 152: 307-316; J. Sambrook et al., “Molecular Cloning: ALaboratory Manual”, 1989, 2nd Ed., Cold Spring Harbour Laboratory Press:New York; “Short Protocols in Molecular Biology”, F. M. Ausubel (Ed.),2002, 5th Ed., John Wiley & Sons; U.S. Pat. Nos. 4,683,195; 4,683,202and 4,800,159). Reverse transcription reactions can be carried out usingnon-specific primers, such as an anchored oligo-dT primer, or randomsequence primers, or using a target-specific primer complementary to theRNA for each probe being monitored, or using thermostable DNApolymerases (such as avian myeloblastosis virus reverse transcriptase orMoloney murine leukemia virus reverse transcriptase).

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the art are intended to be within the scope of the invention.Other embodiments are in the claims.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The followingreferences contain embodiments of the methods and compositions that canbe used herein: The Merck Manual of Diagnosis and Therapy, 18th Edition,published by Merck Research Laboratories, 2006 (ISBN 0-911910-18-2);Benjamin Lewin, Genes IX, published by Jones & Bartlett Publishing, 2007(ISBN-13: 9780763740634); Kendrew et al. (eds.), The Encyclopedia ofMol. Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Mol. Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Standard procedures of the present disclosure are described, e.g., inManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrooket al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis etal., Basic Methods in Molecular Biology, Elsevier Science Publishing,Inc., New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol. 152, S. L. Berger and A. R. Kimmerl (eds.),Academic Press Inc., San Diego, USA (1987)). Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols inImmunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et.al. ed., John Wiley and Sons, Inc.), Culture of Animal Cells: A Manualof Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5thedition (2005), and Animal Cell Culture Methods (Methods in CellBiology, Vol. 57, Jennie P. Mather and David Barnes editors, AcademicPress, 1st edition, 1998), which are all incorporated by referenceherein in their entireties.

It should be understood that the following examples should not beconstrued as being limiting to the particular methodology, protocols,and compositions, etc., described herein and, as such, can vary. Thefollowing terms used herein are for the purpose of describing particularembodiments only, and are not intended to limit the scope of theembodiments disclosed herein.

Disclosed herein are molecules, materials, compositions, and componentsthat can be used for, can be used in conjunction with, can be used inpreparation for, or are products of methods and compositions disclosedherein. It is understood that when combinations, subsets, interactions,groups, etc. of these materials are disclosed and while specificreference of each various individual and collective combinations andpermutation of these molecules and compounds cannot be explicitlydisclosed, each is specifically contemplated and described herein. Forexample, if a nucleotide or nucleic acid is disclosed and discussed anda number of modifications that can be made to a number of moleculesincluding the nucleotide or nucleic acid are discussed, each and everycombination and permutation of nucleotide or nucleic acid and themodifications that are possible are specifically contemplated unlessspecifically indicated to the contrary. This concept applies to allaspects of this application including, but not limited to, steps inmethods of making and using the disclosed molecules and compositions.Thus, if there are a variety of additional steps that can be performedit is understood that each of these additional steps can be performedwith any specific embodiment or combination of embodiments of thedisclosed methods, and that each such combination is specificallycontemplated and should be considered disclosed.

Those skilled in the art can recognize, or be able to ascertain using nomore than routine experimentation, many equivalents to the specificembodiments of the method and compositions described herein. Suchequivalents are intended to be encompassed by the following claims.

It is understood that the disclosed methods and compositions are notlimited to the particular methodology, protocols, and reagents describedas these can vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present disclosure which canbe limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the meanings that would be commonly understood by one of skill inthe art in the context of the present specification.

It should be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “anucleotide” includes a plurality of such nucleotides; reference to “thenucleotide” is a reference to one or more nucleotides and equivalentsthereof known to those skilled in the art, and so forth.

The term “and/or” shall in the present context be understood to indicatethat either or both of the items connected by it are involved. Whilepreferred embodiments of the present disclosure have been shown anddescribed herein, it can be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions can now occur to those skilled inthe art without departing from the disclosure. It should be understoodthat various alternatives to the embodiments of the disclosure describedherein can be employed in practicing the disclosure. It is intended thatthe following claims define the scope of the disclosure and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Sequences:

SEQ ID NO 1: Wild-type A2M precursor protein - complete vector DNAsequence including tag sequences for easier purification.    1CTCATGACCA AAATCCCTTA ACGTGAGTTA CGCGCGCGTC GTTCCACTGA GCGTCAGACC   61CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT  121TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA  181CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTTCTTCTAG  241TGTAGCCGTA GTTAGCCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC  301TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG  361ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA  421CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT  481GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG  541TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTTTATAGTC  601CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG TCAGGGGGGC  661GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC  721CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG  781CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC GAGTCAGTGA  841GCGAGGAAGC GGAAGGCGAG AGTAGGGAAC TGCCAGGCAT CAAACTAAGC AGAAGGCCCC  901TGACGGATGG CCTTTTTGCG TTTCTACAAA CTCTTTCTGT GTTGTAAAAC GACGGCCAGT  961CTTAAGCTCG GGCCCCCTGG GCGGTTCTGA TAACGAGTAA TCGTTAATCC GCAAATAACG 1021TAAAAACCCG CTTCGGCGGG TTTTTTTATG GGGGGAGTTT AGGGAAAGAG CATTTGTCAG 1081AATATTTAAG GGCGCCTGTC ACTTTGCTTG ATATATGAGA ATTATTTAAC CTTATAAATG 1141AGAAAAAAGC AACGCACTTT AAATAAGATA CGTTGCTTTT TCGATTGATG AACACCTATA 1201ATTAAACTAT TCATCTATTA TTTATGATTT TTTGTATATA CAATATTTCT AGTTTGTTAA 1261AGAGAATTAA GAAAATAAAT CTCGAAAATA ATAAAGGGAA AATCAGTTTT TGATATCAAA 1321ATTATACATG TCAACGATAA TACAAAATAT AATACAAACT ATAAGATGTT ATCAGTATTT 1381ATTATCATTT AGAATAAATT TTGTGTCGCC CTTAATTGTG AGCGGATAAC AATTACGAGC 1441TTCATGCACA GTGGCGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA TCAATTACGG 1501GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC 1561CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA 1621TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG 1681CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG 1741ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT 1801GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA 1861TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG 1921TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT 1981CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG 2041CTCTCTGGCT AACTAGAGAA CCCACTGCTT ACTGGCTTAT CGAAATTAAT ACGACTCACT 2101ATAGGGGTAC CTGCCACCAT GGGGAAAAAC AAACTGCTGC ATCCAAGCCT GGTCCTGCTG 2161CTGCTGGTTC TGCTGCCTAC TGACGCCTCT GTGAGCGGAA AGCCCCAGTA TATGGTTCTG 2221GTCCCGTCCC TGCTGCACAC CGAGACCACA GAAAAAGGGT GCGTGCTGCT GTCTTACCTG 2281AATGAAACAG TGACTGTTAG TGCCTCACTG GAGAGTGTGC GCGGAAATCG TTCACTGTTC 2341ACCGATCTGG AGGCGGAAAA CGATGTGCTG CATTGCGTCG CATTTGCTGT GCCAAAAAGC 2401TCCTCTAATG AAGAAGTGAT GTTCCTGACC GTCCAGGTGA AGGGCCCTAC ACAGGAATTC 2461AAAAAACGCA CTACCGTTAT GGTCAAAAAC GAGGATAGCC TGGTGTTTGT TCAGACAGAC 2521AAATCCATCT ATAAGCCTGG TCAGACTGTG AAGTTCCGGG TGGTTAGCAT GGATGAAAAT 2581TTTCACCCCC TGAACGAGCT GATTCCACTG GTGTACATCC AGGACCCTAA AGGCAACCGC 2641ATCGCCCAGT GGCAGTCTTT CCAGCTGGAA GGCGGTCTGA AGCAGTTTAG TTTCCCTCTG 2701AGTTCAGAGC CGTTTCAGGG TTCTTATAAA GTCGTGGTTC AGAAAAAGAG TGGGGGACGT 2761ACTGAACATC CTTTTACCGT TGAAGAGTTC GTCCTGCCGA AATTTGAGGT CCAGGTGACC 2821GTTCCCAAGA TTATCACAAT TCTGGAAGAG GAAATGAACG TGAGCGTGTG CGGACTGTAT 2881ACCTACGGCA AACCAGTGCC TGGTCACGTT ACAGTCAGTA TCTGCCGTAA GTACTCAGAT 2941GCAAGCGACT GTCATGGCGA AGATTCACAG GCTTTTTGCG AGAAGTTCAG CGGCCAGCTG 3001AACTCCCACG GTTGCTTCTA TCAGCAGGTG AAAACCAAGG TTTTTCAGCT GAAACGGAAG 3061GAGTACGAAA TGAAACTGCA TACAGAAGCC CAGATTCAGG AAGAAGGCAC CGTCGTGGAA 3121CTGACTGGTC GTCAGAGCTC CGAGATTACC CGGACAATCA CTAAACTGAG CTTCGTGAAG 3181GTTGATTCCC ACTTTCGGCA GGGGATTCCC TTTTTCGGAC AGGTGCGCCT GGTTGACGGG 3241AAAGGAGTTC CGATCCCCAA CAAAGTGATC TTTATTCGCG GCAATGAAGC CAACTATTAC 3301AGCAACGCGA CAACTGATGA GCATGGGCTG GTGCAGTTCA GTATCAATAC CACAAACGTG 3361ATGGGAACCT CACTGACAGT CCGCGTGAAT TATAAAGACC GTTCACCGTG TTATGGCTAC 3421CAGTGGGTGA GCGAGGAACA CGAGGAAGCC CACCATACCG CGTACCTGGT TTTCAGCCCC 3481TCCAAATCTT TTGTCCATCT GGAACCTATG TCTCACGAGC TGCCGTGCGG CCATACCCAG 3541ACAGTGCAGG CACATTATAT TCTGAACGGC GGCACCCTGC TGGGTCTGAA AAAGCTGAGC 3601TTTTATTACC TGATTATGGC TAAGGGGGGA ATCGTCCGCA CTGGCACCCA CGGTCTGCTG 3661GTTAAACAGG AAGATATGAA GGGCCATTTC AGTATTTCAA TCCCTGTTAA AAGCGACATT 3721GCTCCGGTCG CCCGTCTGCT GATCTATGCC GTGCTGCCAA CCGGCGATGT TATCGGTGAC 3781TCCGCCAAAT ACGATGTGGA GAATTGTCTG GCGAACAAGG TTGACCTGAG CTTTTCCCCC 3841TCTCAGAGTC TGCCAGCGTC TCATGCACAT CTGCGTGTGA CCGCAGCCCC TCAGAGCGTT 3901TGCGCTCTGC GTGCAGTGGA TCAGTCCGTG CTGCTGATGA AGCCAGACGC AGAACTGTCT 3961GCTAGCAGCG TGTATAATCT GCTGCCTGAG AAAGATCTGA CCGGGTTCCC AGGACCTCTG 4021AACGATCAGG ATGACGAAGA CTGTATTAAT CGCCACAACG TGTATATTAA TGGGATCACA 4081TACACTCCGG TTTCAAGCAC CAACGAAAAA GATATGTACA GCTTCCTGGA GGACATGGGT 4141CTGAAAGCGT TTACCAATTC CAAGATCCGG AAACCCAAGA TGTGCCCACA GCTGCAGCAG 4201TATGAAATGC ACGGACCTGA GGGTCTGCGT GTGGGCTTTT ACGAATCTGA TGTGATGGGA 4261CGTGGTCATG CACGTCTGGT TCATGTCGAG GAACCACACA CCGAAACAGT GCGTAAATAC 4321TTCCCTGAGA CCTGGATTTG GGACCTGGTT GTGGTGAACT CCGCGGGTGT GGCAGAAGTG 4381GGTGTTACCG TCCCGGATAC TATTACCGAA TGGAAAGCAG GTGCCTTCTG TCTGTCTGAG 4441GATGCAGGGC TGGGAATCTC CTCTACAGCC TCTCTGCGCG CGTTTCAGCC CTTTTTCGTC 4501GAACTGACTA TGCCATATAG CGTGATTCGT GGCGAGGCAT TCACTCTGAA AGCTACCGTG 4561CTGAATTACC TGCCCAAGTG CATCCGCGTG AGCGTGCAGC TGGAAGCTAG TCCCGCCTTT 4621CTGGCGGTCC CAGTGGAGAA GGAACAGGCA CCGCACTGCA TTTGTGCTAA CGGCCGGCAG 4681ACTGTTTCCT GGGCCGTCAC CCCCAAATCT CTGGGTAATG TGAACTTCAC CGTTTCAGCA 4741GAGGCTCTGG AAAGCCAGGA GCTGTGCGGC ACCGAAGTCC CATCCGTGCC TGAGCATGGT 4801CGCAAAGATA CAGTCATCAA GCCTCTGCTG GTTGAACCGG AAGGCCTGGA GAAGGAAACT 4861ACCTTTAATT CTCTGCTGTG CCCAAGTGGC GGTGAAGTGT CCGAGGAACT GTCTCTGAAA 4921CTGCCGCCCA ACGTGGTCGA GGAATCTGCC CGTGCGTCAG TTAGCGTCCT GGGGGATATT 4981CTGGGAAGTG CCATGCAGAA TACCCAGAAC CTGCTGCAGA TGCCGTATGG CTGTGGCGAG 5041CAGAATATGG TTCTGTTTGC GCCCAACATC TATGTCCTGG ATTACCTGAA TGAAACACAG 5101CAGCTGACTC CTGAAATCAA AAGCAAGGCA ATCGGGTATC TGAATACCGG ATACCAGCGG 5161CAGCTGAACT ATAAGCACTA CGACGGCTCC TATTCTACCT TCGGCGAACG GTACGGTCGC 5221AATCAGGGGA ACACTTGGCT GACCGCCTTT GTGCTGAAAA CCTTTGCCCA GGCTCGCGCC 5281TATATCTTTA TTGATGAGGC CCATATTACA CAGGCGCTGA TCTGGCTGTC ACAGCGCCAG 5341AAGGACAACG GGTGTTTCCG TAGTTCAGGA AGCCTGCTGA ACAATGCCAT CAAAGGCGGC 5401GTCGAGGATG AAGTGACACT GAGCGCATAC ATTACTATCG CTCTGCTGGA AATCCCTCTG 5461ACAGTGACTC ACCCGGTGGT TCGCAATGCT CTGTTTTGCC TGGAAAGTGC ATGGAAAACA 5521GCTCAGGAAG GCGATCACGG ATCACACGTG TATACTAAGG CACTGCTGGC GTACGCATTC 5581GCTCTGGCCG GCAACCAGGA TAAACGTAAA GAAGTGCTGA AATCACTGAA TGAGGAAGCA 5641GTTAAAAAGG ACAACAGCGT CCACTGGGAA CGGCCGCAGA AACCCAAGGC TCCAGTGGGT 5701CACTTTTATG AGCCTCAGGC ACCGAGTGCT GAGGTGGAAA TGACCTCATA TGTTCTGCTG 5761GCATACCTGA CCGCACAGCC TGCCCCCACA TCAGAAGATC TGACAAGCGC CACTAATATT 5821GTGAAATGGA TCACCAAGCA GCAGAACGCG CAGGGCGGTT TTAGCTCCAC CCAGGACACA 5881GTCGTGGCAC TGCACGCTCT GTCTAAATAT GGGGCAGCTA CCTTCACACG CACTGGAAAG 5941GCCGCGCAAG TGACTATTCA GTCTAGTGGC ACCTTTTCAA GCAAGTTCCA GGTGGATAAC 6001AATAACCGTC TGCTGCTGCA GCAGGTGTCC CTGCCCGAAC TGCCAGGCGA GTACTCTATG 6061AAAGTCACTG GGGAAGGATG CGTGTATCTG CAGACCTCCC TGAAATACAA TATTCTGCCC 6121GAGAAAGAAG AATTTCCATT CGCACTGGGC GTGCAGACCC TGCCTCAGAC ATGCGATGAA 6181CCGAAGGCTC ATACTTCTTT TCAGATCAGT CTGTCAGTGA GCTATACCGG GTCCCGCTCT 6241GCCAGTAACA TGGCGATTGT GGATGTGAAA ATGGTGAGTG GATTCATCCC TCTGAAACCG 6301ACTGTGAAGA TGCTGGAACG GAGTAATCAC GTTTCACGCA CCGAGGTCTC CTCTAACCAT 6361GTGCTGATCT ACCTGGATAA AGTGTCCAAT CAGACACTGT CTCTGTTTTT CACTGTGCTG 6421CAGGATGTCC CCGTGCGTGA CCTGAAACCA GCCATTGTTA AGGTCTATGA TTATTACGAA 6481ACCGACGAGT TCGCGATCGC AGAATACAAC GCGCCGTGCA GCAAAGACCT GGGGAATGCT 6541GACTACAAGG ACGACGACGA CAAGGGGGCA AGCCACCACC ATCACCATCA CTAAGGATCC 6601AAAATCAGCC TCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC CCTCCCCCGT 6661GCCTTCCTTG ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA ATGAGGAAAT 6721TGCATCACAA CACTCAACCC TATCTCGGTC TATTCTTTTG ATTTATAAGG GATTTTGCCG 6781ATTTCGGCCT ATTGGTTAAA AAATGAGCTG ATTTAACAAA AATTTAACGC GAATTAATTC 6841TGTGGAATGT GTGTCAGTTA GGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA 6901TGCAAAGCAT GCATCTCAAT TAGTCAGCAA CCAGGTGTGG AAAGTCCCCA GGCTCCCCAG 6961CAGGCAGAAG TATGCAAAGC ATGCATCTCA ATTAGTCAGC AACCATAGTC CCGCCCCTAA 7021CTCCGCCCAT CCCGCCCCTA ACTCCGCCCA GTTCCGCCCA TTCTCCGCCC CATGGCTGAC 7081TAATTTTTTT TATTTATGCA GAGGCCGAGG CCGCCTCTGC CTCTGAGCTA TTCCAGAAGT 7141AGTGAGGAGG CTTTTTTGGA GGCCTAGGCT TTTGCAAAAA GCTCCCGGGA GCTTGTATAT 7201CCATTTTCGG ATCTGATCAG CACGTGTTGA CAATTAATCA TCGGCATAGT ATATCGGCAT 7261AGTATAATAC GACAAGGTGA GGAACTAAAC CATGGCCAAG CCTTTGTCTC AAGAAGAATC 7321CACCCTCATT GAAAGAGCAA CGGCTACAAT CAACAGCATC CCCATCTCTG AAGACTACAG 7381CGTCGCCAGC GCAGCTCTCT CTAGCGACGG CCGCATCTTC ACTGGTGTCA ATGTATATCA 7441TTTTACTGGG GGACCTTGTG CAGAACTCGT GGTGCTGGGC ACTGCTGCTG CTGCGGCAGC 7501TGGCAACCTG ACTTGTATCG TCGCGATCGG AAATGAGAAC AGGGGCATCT TGAGCCCCTG 7561CGGACGGTGC CGACAGGTGC TTCTCGATCT GCATCCTGGG ATCAAAGCCA TAGTGAAGGA 7621CAGTGATGGA CAGCCGACGG CAGTTGGGAT TCGTGAATTG CTGCCCTCTG GTTATGTGTG 7681GGAGGGCTAA CACGTGCTAC GAGATTTCGA TTCCACCGCC GCCTTCTATG AAAGGTTGGG 7741CTTCGGAATC GTTTTCCGGG ACGCCGGCTG GATGATCCTC CAGCGCGGGG ATCTCATGCT 7801GGAGTTCTTC GCCCACCCCA ACTTGTTTAT TGCAGCTTAT AATGGTTACA AATAAAGCAA 7861TAGCATCACA AATTTCACAA ATAAAGCATT TTTTTCACTG CATTCTAGTT GTGGTTTGTC 7921CAAACTCATC AATGTATCTT ATCATGTCTG TATACCGTCG ACCTCTAGCT AGAGCTTGGC 7981GTAATCATGG TCATTACCAA TGCTTAATCA GTGAGGCACC TATCTCAGCG ATCTGTCTAT 8041TTCGTTCATC CATAGTTGCC TGACTCCCCG TCGTGTAGAT AACTACGATA CGGGAGGGCT 8101TACCATCTGG CCCCAGCGCT GCGATGATAC CGCGAGAACC ACGCTCACCG GCTCCGGATT 8161TATCAGCAAT AAACCAGCCA GCCGGAAGGG CCGAGCGCAG AAGTGGTCCT GCAACTTTAT 8221CCGCCTCCAT CCAGTCTATT AATTGTTGCC GGGAAGCTAG AGTAAGTAGT TCGCCAGTTA 8281ATAGTTTGCG CAACGTTGTT GCCATCGCTA CAGGCATCGT GGTGTCACGC TCGTCGTTTG 8341GTATGGCTTC ATTCAGCTCC GGTTCCCAAC GATCAAGGCG AGTTACATGA TCCCCCATGT 8401TGTGCAAAAA AGCGGTTAGC TCCTTCGGTC CTCCGATCGT TGTCAGAAGT AAGTTGGCCG 8461CAGTGTTATC ACTCATGGTT ATGGCAGCAC TGCATAATTC TCTTACTGTC ATGCCATCCG 8521TAAGATGCTT TTCTGTGACT GGTGAGTACT CAACCAAGTC ATTCTGAGAA TAGTGTATGC 8581GGCGACCGAG TTGCTCTTGC CCGGCGTCAA TACGGGATAA TACCGCGCCA CATAGCAGAA 8641CTTTAAAAGT GCTCATCATT GGAAAACGTT CTTCGGGGCG AAAACTCTCA AGGATCTTAC 8701CGCTGTTGAG ATCCAGTTCG ATGTAACCCA CTCGTGCACC CAACTGATCT TCAGCATCTT 8761TTACTTTCAC CAGCGTTTCT GGGTGAGCAA AAACAGGAAG GCAAAATGCC GCAAAAAAGG 8821GAATAAGGGC GACACGGAAA TGTTGAATAC TCATATTCTT CCTTTTTCAA TATTATTGAA 8881GCATTTATCA GGGTTATTGT CTCATGAGCG GATACATATT TGAATGTATT TAGAAAAATA 8941AACAAATAGG GGTCAGTGTT ACAACCAATT AACCAATTCT GAACATTATC GCGSEQ ID NO 2: Complete vector DNA sequence of the of the acceptor mutant.   1 CTCATGACCA AAATCCCTTA ACGTGAGTTA CGCGCGCGTC GTTCCACTGA GCGTCAGACC  61 CCGTAGAAAA GATCAAAGGA TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT 121 TGCAAACAAA AAAACCACCG CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA 181 CTCTTTTTCC GAAGGTAACT GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTTCTTCTAG 241 TGTAGCCGTA GTTAGCCCAC CACTTCAAGA ACTCTGTAGC ACCGCCTACA TACCTCGCTC 301 TGCTAATCCT GTTACCAGTG GCTGCTGCCA GTGGCGATAA GTCGTGTCTT ACCGGGTTGG 361 ACTCAAGACG ATAGTTACCG GATAAGGCGC AGCGGTCGGG CTGAACGGGG GGTTCGTGCA 421 CACAGCCCAG CTTGGAGCGA ACGACCTACA CCGAACTGAG ATACCTACAG CGTGAGCTAT 481 GAGAAAGCGC CACGCTTCCC GAAGGGAGAA AGGCGGACAG GTATCCGGTA AGCGGCAGGG 541 TCGGAACAGG AGAGCGCACG AGGGAGCTTC CAGGGGGAAA CGCCTGGTAT CTTTATAGTC 601 CTGTCGGGTT TCGCCACCTC TGACTTGAGC GTCGATTTTT GTGATGCTCG TCAGGGGGGC 661 GGAGCCTATG GAAAAACGCC AGCAACGCGG CCTTTTTACG GTTCCTGGCC TTTTGCTGGC 721 CTTTTGCTCA CATGTTCTTT CCTGCGTTAT CCCCTGATTC TGTGGATAAC CGTATTACCG 781 CCTTTGAGTG AGCTGATACC GCTCGCCGCA GCCGAACGAC CGAGCGCAGC GAGTCAGTGA 841 GCGAGGAAGC GGAAGGCGAG AGTAGGGAAC TGCCAGGCAT CAAACTAAGC AGAAGGCCCC 901 TGACGGATGG CCTTTTTGCG TTTCTACAAA CTCTTTCTGT GTTGTAAAAC GACGGCCAGT 961 CTTAAGCTCG GGCCCCCTGG GCGGTTCTGA TAACGAGTAA TCGTTAATCC GCAAATAACG1021 TAAAAACCCG CTTCGGCGGG TTTTTTTATG GGGGGAGTTT AGGGAAAGAG CATTTGTCAG1081 AATATTTAAG GGCGCCTGTC ACTTTGCTTG ATATATGAGA ATTATTTAAC CTTATAAATG1141 AGAAAAAAGC AACGCACTTT AAATAAGATA CGTTGCTTTT TCGATTGATG AACACCTATA1201 ATTAAACTAT TCATCTATTA TTTATGATTT TTTGTATATA CAATATTTCT AGTTTGTTAA1261 AGAGAATTAA GAAAATAAAT CTCGAAAATA ATAAAGGGAA AATCAGTTTT TGATATCAAA1321 ATTATACATG TCAACGATAA TACAAAATAT AATACAAACT ATAAGATGTT ATCAGTATTT1381 ATTATCATTT AGAATAAATT TTGTGTCGCC CTTAATTGTG AGCGGATAAC AATTACGAGC1441 TTCATGCACA GTGGCGTTGA CATTGATTAT TGACTAGTTA TTAATAGTAA TCAATTACGG1501 GGTCATTAGT TCATAGCCCA TATATGGAGT TCCGCGTTAC ATAACTTACG GTAAATGGCC1561 CGCCTGGCTG ACCGCCCAAC GACCCCCGCC CATTGACGTC AATAATGACG TATGTTCCCA1621 TAGTAACGCC AATAGGGACT TTCCATTGAC GTCAATGGGT GGAGTATTTA CGGTAAACTG1681 CCCACTTGGC AGTACATCAA GTGTATCATA TGCCAAGTAC GCCCCCTATT GACGTCAATG1741 ACGGTAAATG GCCCGCCTGG CATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT1801 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA1861 TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG1921 TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT1981 CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG2041 CTCTCTGGCT AACTAGAGAA CCCACTGCTT ACTGGCTTAT CGAAATTAAT ACGACTCACT2101 ATAGGGGTAC CTGCCACCAT GGGGAAAAAC AAACTGCTGC ATCCAAGCCT GGTCCTGCTG2161 CTGCTGGTTC TGCTGCCTAC TGACGCCTCT GTGAGCGGAA AGCCCCAGTA TATGGTTCTG2221 GTCCCGTCCC TGCTGCACAC CGAGACCACA GAAAAAGGGT GCGTGCTGCT GTCTTACCTG2281 AATGAAACAG TGACTGTTAG TGCCTCACTG GAGAGTGTGC GCGGAAATCG TTCACTGTTC2341 ACCGATCTGG AGGCGGAAAA CGATGTGCTG CATTGCGTCG CATTTGCTGT GCCAAAAAGC2401 TCCTCTAATG AAGAAGTGAT GTTCCTGACC GTCCAGGTGA AGGGCCCTAC ACAGGAATTC2461 AAAAAACGCA CTACCGTTAT GGTCAAAAAC GAGGATAGCC TGGTGTTTGT TCAGACAGAC2521 AAATCCATCT ATAAGCCTGG TCAGACTGTG AAGTTCCGGG TGGTTAGCAT GGATGAAAAT2581 TTTCACCCCC TGAACGAGCT GATTCCACTG GTGTACATCC AGGACCCTAA AGGCAACCGC2641 ATCGCCCAGT GGCAGTCTTT CCAGCTGGAA GGCGGTCTGA AGCAGTTTAG TTTCCCTCTG2701 AGTTCAGAGC CGTTTCAGGG TTCTTATAAA GTCGTGGTTC AGAAAAAGAG TGGGGGACGT2761 ACTGAACATC CTTTTACCGT TGAAGAGTTC GTCCTGCCGA AATTTGAGGT CCAGGTGACC2821 GTTCCCAAGA TTATCACAAT TCTGGAAGAG GAAATGAACG TGAGCGTGTG CGGACTGTAT2881 ACCTACGGCA AACCAGTGCC TGGTCACGTT ACAGTCAGTA TCTGCCGTAA GTACTCAGAT2941 GCAAGCGACT GTCATGGCGA AGATTCACAG GCTTTTTGCG AGAAGTTCAG CGGCCAGCTG3001 AACTCCCACG GTTGCTTCTA TCAGCAGGTG AAAACCAAGG TTTTTCAGCT GAAACGGAAG3061 GAGTACGAAA TGAAACTGCA TACAGAAGCC CAGATTCAGG AAGAAGGCAC CGTCGTGGAA3121 CTGACTGGTC GTCAGAGCTC CGAGATTACC CGGACAATCA CTAAACTGAG CTTCGTGAAG3181 GTTGATTCCC ACTTTCGGCA GGGGATTCCC TTTTTCGGAC AGGTGCGCCT GGTTGACGGG3241 AAAGGAGTTC CGATCCCCAA CAAAGTGATC TTTATTCGCG GCAATGAAGC CAACTATTAC3301 AGCAACGCGA CAACTGATGA GCATGGGCTG GTGCAGTTCA GTATCAATAC CACAAACGTG3361 ATGGGAACCT CACTGACAGT CCGCGTGAAT TATAAAGACC GTTCACCGTG TTATGGCTAC3421 CAGTGGGTGA GCGAGGAACA CGAGGAAGCC CACCATACCG CGTACCTGGT TTTCAGCCCC3481 TCCAAATCTT TTGTCCATCT GGAACCTATG TCTCACGAGC TGCCGTGCGG CCATACCCAG3541 ACAGTGCAGG CACATTATAT TCTGAACGGC GGCACCCTGC TGGGTCTGAA AAAGCTGAGC3601 TTTTATTACC TGATTATGGC TAAGGGGGGA ATCGTCCGCA CTGGCACCCA CGGTCTGCTG3661 GTTAAACAGG AAGATATGAA GGGCCATTTC AGTATTTCAA TCCCTGTTAA AAGCGACATT3721 GCTCCGGTCG CCCGTCTGCT GATCTATGCC GTGCTGCCAA CCGGCGATGT TATCGGTGAC3781 TCCGCCAAAT ACGATGTGGA GAATTGTCTG GCGAACAAGG TTGACCTGAG CTTTTCCCCC3841 TCTCAGAGTC TGCCAGCGTC TCATGCACAT CTGCGTGTGA CCGCAGCCCC TCAGAGCGTT3901 TGCGCTCTGC GTGCAGTGGA TCAGTCCGTG CTGCTGATGA AGCCAGACGC AGAACTGTCT3961 GCTAGCAGCG TGTATAATCT GCTGCCTGAG AAAGATCTGA CCGGGTTCCC AGGACCTCTG4021 AACGATCAGG ATGACGAAGA CTGTATTAAT CGCCACAACG TGTATATTAA TGGGATCACA4081 TACACTCCGG TTTCAAGCAC CAACGAAAAA GATATGTACA GCTTCCTGGA GGACATGGGT4141 CTGAAAGCGT TTACCAATTC CAAGATCCGG AAACCCCAAG ATGTGCCCAC AGCTCGAGCA4201 GTATGAAATG CACGGACCTG AGGGTCTGCG TGTGGGCTTT TACGAATCTG ATGTGATGGG4261 ACGTGGTCAT GCACGTCTGG TTCATGTCGA GGAACCACAC ACCGAAAAGC TTCGTAAATA4321 CTTCCCTGAG ACCTGGATTT GGGACCTGGT TGTGGTGAAC TCCGCGGGTG TGGCAGAAGT4381 GGGTGTTACC GTCCCGGATA CTATTACCGA ATGGAAAGCA GGTGCCTTCT GTCTGTCTGA4441 GGATGCAGGG CTGGGAATCT CCTCTACAGC CTCTCTGCGC GCGTTTCAGC CCTTTTTCGT4501 CGAACTGACT ATGCCATATA GCGTGATTCG TGGCGAGGCA TTCACTCTGA AAGCTACCGT4561 GCTGAATTAC CTGCCCAAGT GCATCCGCGT GAGCGTGCAG CTGGAAGCTA GTCCCGCCTT4621 TCTGGCGGTC CCAGTGGAGA AGGAACAGGC ACCGCACTGC ATTTGTGCTA ACGGCCGGCA4681 GACTGTTTCC TGGGCCGTCA CCCCCAAATC TCTGGGTAAT GTGAACTTCA CCGTTTCAGC4741 AGAGGCTCTG GAAAGCCAGG AGCTGTGCGG CACCGAAGTC CCATCCGTGC CTGAGCATGG4801 TCGCAAAGAT ACAGTCATCA AGCCTCTGCT GGTTGAACCG GAAGGCCTGG AGAAGGAAAC4861 TACCTTTAAT TCTCTGCTGT GCCCAAGTGG CGGTGAAGTG TCCGAGGAAC TGTCTCTGAA4921 ACTGCCGCCC AACGTGGTCG AGGAATCTGC CCGTGCGTCA GTTAGCGTCC TGGGGGATAT4981 TCTGGGAAGT GCCATGCAGA ATACCCAGAA CCTGCTGCAG ATGCCGTATG GCTGTGGCGA5041 GCAGAATATG GTTCTGTTTG CGCCCAACAT CTATGTCCTG GATTACCTGA ATGAAACACA5101 GCAGCTGACT CCTGAAATCA AAAGCAAGGC AATCGGGTAT CTGAATACCG GATACCAGCG5161 GCAGCTGAAC TATAAGCACT ACGACGGCTC CTATTCTACC TTCGGCGAAC GGTACGGTCG5221 CAATCAGGGG AACACTTGGC TGACCGCCTT TGTGCTGAAA ACCTTTGCCC AGGCTCGCGC5281 CTATATCTTT ATTGATGAGG CCCATATTAC ACAGGCGCTG ATCTGGCTGT CACAGCGCCA5341 GAAGGACAAC GGGTGTTTCC GTAGTTCAGG AAGCCTGCTG AACAATGCCA TCAAAGGCGG5401 CGTCGAGGAT GAAGTGACAC TGAGCGCATA CATTACTATC GCTCTGCTGG AAATCCCTCT5461 GACAGTGACT CACCCGGTGG TTCGCAATGC TCTGTTTTGC CTGGAAAGTG CATGGAAAAC5521 AGCTCAGGAA GGCGATCACG GATCACACGT GTATACTAAG GCACTGCTGG CGTACGCATT5581 CGCTCTGGCC GGCAACCAGG ATAAACGTAA AGAAGTGCTG AAATCACTGA ATGAGGAAGC5641 AGTTAAAAAG GACAACAGCG TCCACTGGGA ACGGCCGCAG AAACCCAAGG CTCCAGTGGG5701 TCACTTTTAT GAGCCTCAGG CACCGAGTGC TGAGGTGGAA ATGACCTCAT ATGTTCTGCT5761 GGCATACCTG ACCGCACAGC CTGCCCCCAC ATCAGAAGAT CTGACAAGCG CCACTAATAT5821 TGTGAAATGG ATCACCAAGC AGCAGAACGC GCAGGGCGGT TTTAGCTCCA CCCAGGACAC5881 AGTCGTGGCA CTGCACGCTC TGTCTAAATA TGGGGCAGCT ACCTTCACAC GCACTGGAAA5941 GGCCGCGCAA GTGACTATTC AGTCTAGTGG CACCTTTTCA AGCAAGTTCC AGGTGGATAA6001 CAATAACCGT CTGCTGCTGC AGCAGGTGTC CCTGCCCGAA CTGCCAGGCG AGTACTCTAT6061 GAAAGTCACT GGGGAAGGAT GCGTGTATCT GCAGACCTCC CTGAAATACA ATATTCTGCC6121 CGAGAAAGAA GAATTTCCAT TCGCACTGGG CGTGCAGACC CTGCCTCAGA CATGCGATGA6181 ACCGAAGGCT CATACTTCTT TTCAGATCAG TCTGTCAGTG AGCTATACCG GGTCCCGCTC6241 TGCCAGTAAC ATGGCGATTG TGGATGTGAA AATGGTGAGT GGATTCATCC CTCTGAAACC6301 GACTGTGAAG ATGCTGGAAC GGAGTAATCA CGTTTCACGC ACCGAGGTCT CCTCTAACCA6361 TGTGCTGATC TACCTGGATA AAGTGTCCAA TCAGACACTG TCTCTGTTTT TCACTGTGCT6421 GCAGGATGTC CCCGTGCGTG ACCTGAAACC AGCCATTGTT AAGGTCTATG ATTATTACGA6481 AACCGACGAG TTCGCGATCG CAGAATACAA CGCGCCGTGC AGCAAAGACC TGGGGAATGC6541 TGACTACAAG GACGACGACG ACAAGGGGGC AAGCCACCAC CATCACCATC ACTAAGGATC6601 CAAAATCAGC CTCGACTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC CCCTCCCCCG6661 TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA AATGAGGAAA6721 TTGCATCACA ACACTCAACC CTATCTCGGT CTATTCTTTT GATTTATAAG GGATTTTGCC6781 GATTTCGGCC TATTGGTTAA AAAATGAGCT GATTTAACAA AAATTTAACG CGAATTAATT6841 CTGTGGAATG TGTGTCAGTT AGGGTGTGGA AAGTCCCCAG GCTCCCCAGC AGGCAGAAGT6901 ATGCAAAGCA TGCATCTCAA TTAGTCAGCA ACCAGGTGTG GAAAGTCCCC AGGCTCCCCA6961 GCAGGCAGAA GTATGCAAAG CATGCATCTC AATTAGTCAG CAACCATAGT CCCGCCCCTA7021 ACTCCGCCCA TCCCGCCCCT AACTCCGCCC AGTTCCGCCC ATTCTCCGCC CCATGGCTGA7081 CTAATTTTTT TTATTTATGC AGAGGCCGAG GCCGCCTCTG CCTCTGAGCT ATTCCAGAAG7141 TAGTGAGGAG GCTTTTTTGG AGGCCTAGGC TTTTGCAAAA AGCTCCCGGG AGCTTGTATA7201 TCCATTTTCG GATCTGATCA GCACGTGTTG ACAATTAATC ATCGGCATAG TATATCGGCA7261 TAGTATAATA CGACAAGGTG AGGAACTAAA CCATGGCCAA GCCTTTGTCT CAAGAAGAAT7321 CCACCCTCAT TGAAAGAGCA ACGGCTACAA TCAACAGCAT CCCCATCTCT GAAGACTACA7381 GCGTCGCCAG CGCAGCTCTC TCTAGCGACG GCCGCATCTT CACTGGTGTC AATGTATATC7441 ATTTTACTGG GGGACCTTGT GCAGAACTCG TGGTGCTGGG CACTGCTGCT GCTGCGGCAG7501 CTGGCAACCT GACTTGTATC GTCGCGATCG GAAATGAGAA CAGGGGCATC TTGAGCCCCT7561 GCGGACGGTG CCGACAGGTG CTTCTCGATC TGCATCCTGG GATCAAAGCC ATAGTGAAGG7621 ACAGTGATGG ACAGCCGACG GCAGTTGGGA TTCGTGAATT GCTGCCCTCT GGTTATGTGT7681 GGGAGGGCTA ACACGTGCTA CGAGATTTCG ATTCCACCGC CGCCTTCTAT GAAAGGTTGG7741 GCTTCGGAAT CGTTTTCCGG GACGCCGGCT GGATGATCCT CCAGCGCGGG GATCTCATGC7801 TGGAGTTCTT CGCCCACCCC AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA7861 ATAGCATCAC AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT7921 CCAAACTCAT CAATGTATCT TATCATGTCT GTATACCGTC GACCTCTAGC TAGAGCTTGG7981 CGTAATCATG GTCATTACCA ATGCTTAATC AGTGAGGCAC CTATCTCAGC GATCTGTCTA8041 TTTCGTTCAT CCATAGTTGC CTGACTCCCC GTCGTGTAGA TAACTACGAT ACGGGAGGGC8101 TTACCATCTG GCCCCAGCGC TGCGATGATA CCGCGAGAAC CACGCTCACC GGCTCCGGAT8161 TTATCAGCAA TAAACCAGCC AGCCGGAAGG GCCGAGCGCA GAAGTGGTCC TGCAACTTTA8221 TCCGCCTCCA TCCAGTCTAT TAATTGTTGC CGGGAAGCTA GAGTAAGTAG TTCGCCAGTT8281 AATAGTTTGC GCAACGTTGT TGCCATCGCT ACAGGCATCG TGGTGTCACG CTCGTCGTTT8341 GGTATGGCTT CATTCAGCTC CGGTTCCCAA CGATCAAGGC GAGTTACATG ATCCCCCATG8401 TTGTGCAAAA AAGCGGTTAG CTCCTTCGGT CCTCCGATCG TTGTCAGAAG TAAGTTGGCC8461 GCAGTGTTAT CACTCATGGT TATGGCAGCA CTGCATAATT CTCTTACTGT CATGCCATCC8521 GTAAGATGCT TTTCTGTGAC TGGTGAGTAC TCAACCAAGT CATTCTGAGA ATAGTGTATG8581 CGGCGACCGA GTTGCTCTTG CCCGGCGTCA ATACGGGATA ATACCGCGCC ACATAGCAGA8641 ACTTTAAAAG TGCTCATCAT TGGAAAACGT TCTTCGGGGC GAAAACTCTC AAGGATCTTA8701 CCGCTGTTGA GATCCAGTTC GATGTAACCC ACTCGTGCAC CCAACTGATC TTCAGCATCT8761 TTTACTTTCA CCAGCGTTTC TGGGTGAGCA AAAACAGGAA GGCAAAATGC CGCAAAAAAG8821 GGAATAAGGG CGACACGGAA ATGTTGAATA CTCATATTCT TCCTTTTTCA ATATTATTGA8881 AGCATTTATC AGGGTTATTG TCTCATGAGC GGATACATAT TTGAATGTAT TTAGAAAAAT8941 AAACAAATAG GGGTCAGTGT TACAACCAAT TAACCAATTC TGAACATTAT CGCGSEQ ID NO 3: Amino Acid Sequence of Tagged wild-type human A2M    1MGKNKLLHPS LVLLLLVLLP TDASVSGKPQ YMVLVPSLLH TETTEKGCVL LSYLNETVTV   61SASLESVRGN RSLFTDLEAE NDVLHCVAFA VPKSSSNEEV MFLTVQVKGP TQEFKKRTTV  121MVKNEDSLVF VQTDKSIYKP GQTVKFRVVS MDENFHPLNE LIPLVYIQDP KGNRIAQWQS  181FQLEGGLKQF SFPLSSEPFQ GSYKVVVQKK SGGRTEHPFT VEEFVLPKFE VQVTVPKIIT  241ILEEEMNVSV CGLYTYGKPV PGHVTVSICR KYSDASDCHG EDSQAFCEKF SGQLNSHGCF  301YQQVKTKVFQ LKRKEYEMKL HTEAQIQEEG TVVELTGRQS SEITRTITKL SFVKVDSHFR  361QGIPFFGQVR LVDGKGVPIP NKVIFIRGNE ANYYSNATTD EHGLVQFSIN TTNVMGTSLT  421VRVNYKDRSP CYGYQWVSEE HEEAHHTAYL VFSPSKSFVH LEPMSHELPC GHTQTVQAHY  481ILNGGTLLGL KKLSFYYLIM AKGGIVRTGT HGLLVKQEDM KGHFSISIPV KSDIAPVARL  541LIYAVLPTGD VIGDSAKYDV ENCLANKVDL SFSPSQSLPA SHAHLRVTAA PQSVCALRAV  601DQSVLLMKPD AELSASSVYN LLPEKDLTGF PGPLNDQDDE DCINRHNVYI NGITYTPVSS  661TNEKDMYSFL EDMGLKAFTN SKIRKPKMCP QLQQYEMHGP EGLRVGFYES DVMGRGHARL  721VHVEEPHTET VRKYFPETWI WDLVVVNSAG VAEVGVTVPD TITEWKAGAF CLSEDAGLGI  781SSTASLRAFQ PFFVELTMPY SVIRGEAFTL KATVLNYLPK CIRVSVQLEA SPAFLAVPVE  841KEQAPHCICA NGRQTVSWAV TPKSLGNVNF TVSAEALESQ ELCGTEVPSV PEHGRKDTVI  901KPLLVEPEGL EKETTFNSLL CPSGGEVSEE LSLKLPPNVV EESARASVSV LGDILGSAMQ  961NTQNLLQMPY GCGEQNMVLF APNIYVLDYL NETQQLTPEI KSKAIGYLNT GYQRQLNYKH 1021YDGSYSTFGE RYGRNQGNTW LTAFVLKTFA QARAYIFIDE AHITQALIWL SQRQKDNGCF 1081RSSGSLLNNA IKGGVEDEVT LSAYITIALL EIPLTVTHPV VRNALFCLES AWKTAQEGDH 1141GSHVYTKALL AYAFALAGNQ DKRKEVLKSL NEEAVKKDNS VHWERPQKPK APVGHFYEPQ 1201APSAEVEMTS YVLLAYLTAQ PAPTSEDLTS ATNIVKWITK QQNAQGGFSS TQDTVVALHA 1261LSKYGAATFT RTGKAAQVTI QSSGTFSSKF QVDNNNRLLL QQVSLPELPG EYSMKVTGEG 1321CVYLQTSLKY NILPEKEEFP FALGVQTLPQ TCDEPKAHTS FQISLSVSYT GSRSASNMAI 1381VDVKMVSGFI PLKPTVKMLE RSNHVSRTEV SSNHVLIYLD KVSNQTLSLF FTVLQDVPVR 1441DLKPAIVKVY DYYETDEFAI AEYNAPCSKD LGNADYKDDD DKGASHHHHHHSEQ ID NO 4: Amino Acid Sequence of the Acceptor Mutant.    1MGKNKLLHPS LVLLLLVLLP TDASVSGKPQ YMVLVPSLLH TETTEKGCVL LSYLNETVTV   61SASLESVRGN RSLFTDLEAE NDVLHCVAFA VPKSSSNEEV MFLTVQVKGP TQEFKKRTTV  121MVKNEDSLVF VQTDKSIYKP GQTVKFRVVS MDENFHPLNE LIPLVYIQDP KGNRIAQWQS  181FQLEGGLKQF SFPLSSEPFQ GSYKVVVQKK SGGRTEHPFT VEEFVLPKFE VQVTVPKIIT  241ILEEEMNVSV CGLYTYGKPV PGHVTVSICR KYSDASDCHG EDSQAFCEKF SGQLNSHGCF  301YQQVKTKVFQ LKRKEYEMKL HTEAQIQEEG TVVELTGRQS SEITRTITKL SFVKVDSHFR  361QGIPFFGQVR LVDGKGVPIP NKVIFIRGNE ANYYSNATTD EHGLVQFSIN TTNVMGTSLT  421VRVNYKDRSP CYGYQWVSEE HEEAHHTAYL VFSPSKSFVH LEPMSHELPC GHTQTVQAHY  481ILNGGTLLGL KKLSFYYLIM AKGGIVRTGT HGLLVKQEDM KGHFSISIPV KSDIAPVARL  541LIYAVLPTGD VIGDSAKYDV ENCLANKVDL SFSPSQSLPA SHAHLRVTAA PQSVCALRAV  601DQSVLLMKPD AELSASSVYN LLPEKDLTGF PGPLNDQDDE DCINRHNVYI NGITYTPVSS  661TNEKDMYSFL EDMGLKAFTN SKIRKPKMCP QLEQYEMHGP EGLRVGFYES DVMGRGHARL  721VHVEEPHTEK LRKYFPETWI WDLVVVNSAG VAEVGVTVPD TITEWKAGAF CLSEDAGLGI  781SSTASLRAFQ PFFVELTMPY SVIRGEAFTL KATVLNYLPK CIRVSVQLEA SPAFLAVPVE  841KEQAPHCICA NGRQTVSWAV TPKSLGNVNF TVSAEALESQ ELCGTEVPSV PEHGRKDTVI  901KPLLVEPEGL EKETTFNSLL CPSGGEVSEE LSLKLPPNVV EESARASVSV LGDILGSAMQ  961NTQNLLQMPY GCGEQNMVLF APNIYVLDYL NETQQLTPEI KSKAIGYLNT GYQRQLNYKH 1021YDGSYSTFGE RYGRNQGNTW LTAFVLKTFA QARAYIFIDE AHITQALIWL SQRQKDNGCF 1081RSSGSLLNNA IKGGVEDEVT LSAYITIALL EIPLTVTHPV VRNALFCLES AWKTAQEGDH 1141GSHVYTKALL AYAFALAGNQ DKRKEVLKSL NEEAVKKDNS VHWERPQKPK APVGHFYEPQ 1201APSAEVEMTS YVLLAYLTAQ PAPTSEDLTS ATNIVKWITK QQNAQGGFSS TQDTVVALHA 1261LSKYGAATFT RTGKAAQVTI QSSGTFSSKF QVDNNNRLLL QQVSLPELPG EYSMKVTGEG 1321CVYLQTSLKY NILPEKEEFP FALGVQTLPQ TCDEPKAHTS FQISLSVSYT GSRSASNMAI 1381VDVKMVSGFI PLKPTVKMLE RSNHVSRTEV SSNHVLIYLD KVSNQTLSLF FTVLQDVPVR 1441DLKPAIVKVY DYYETDEFAI AEYNAPCSKD LGNADYKDDD DKGASHHHHH HSEQ ID NO 5: Amino Acid Sequence of wild-type A2M Bait Region.SEQ ID NO: 5 - PQLQQYEMHGPEGLRVGFYESDVMGRGHARLVHVEEPHTETSEQ ID NOs 6-30: Amino Acid Sequences of Variant Bait Regions.SEQ ID NO: 6 - LEHGPEGEGEGEGIPENFYGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 7 - LEHGPEGEGEGEGIPENFFGVRYSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 8 - LEHGPEGEGEGEGIPENFFGVLYSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 9 - LEHGPEGEGEGEGIPENFFGVPRYLSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 10 - LEHGPEGEGLGEGIPENFYGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 11 - LEHGPEGEGEGPRYLTAIPENFFGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 12 - LEHGPEGEGEGEIPENFEFRGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 13 - LEHGPRYLTAEGEGEGIPENFFGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 14 - LEHGPEGEGEGEGIPRYLTAENFFGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 15 - LEHGPEGEGEGEGIPENFFGVSEDLVVQISELEGRGSRYLTAVEEPHTKLSEQ ID NO: 16 - LEHGPEFRGVTRYLTAIPENFYGVSELEGRGSSEDLVVQIVEEPHTKLSEQ ID NO: 17 - LEHGPTEGEARGSIPENFYGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 18 - LEHGPIPENFYGLEGEGEGEGEAIPMSIPRYLTAEFRGVTVEEPHTKLSEQ ID NO: 19 - LEHGPEGEGEGEFRGVTIPENFYGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 20 - LEHGPEFRGVTEGEGEGIPENFYGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 21 - LEHGPTEGEARGSPRYLTAIPENFYGVSEDLVVQISELEGRGSPHTKLSEQ ID NO: 22 - LEHGPEGEGEGEFRGVTIPENFFGVPRYLTASEDLVVQISELEGRGSPHTKLSEQ ID NO: 23 - LEHGPIPENFYGVEGEGLGIGSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 24 - LEHGPIPENFYGVEGEGEGEGSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 25 - LEHGPEGEGEGEGIPENFYGVSEDLYTASELEGRGSVEEPHTKLSEQ ID NO: 26 - LEHGPEGEGEGEFRAAPFLTAIPENFFGVSEDLVVQISELEGRGSPHTKLSEQ ID NO: 27 - LEQYEMHGPEGEGEGEGIPENFYGVSEDLYTASELEGRGSVEEPHTKLSEQ ID NO: 28 - LEQYEMHGPEGEGEGEFRAAPFLTAIPENFFGVSEDLVVQISELEGRGSPHTKLSEQ ID NO: 29 - LEQPEGEGEGPRYLTAIPENFFGVSEDLVVQISELEGRGSVEEPHTKLSEQ ID NO: 30 - LEQYEMHGPEGEGEGPRYLTAIPENFFGVSEDLVVQISELEGRGSPHTKLSEQ ID NOs 31-83: Amino Acid Sequences of Protease Recognition Sites andConsensus Protease Recognition Sites of Variant A2M Bait RegionsSEQ ID NO: 31 - TAQEAGEG SEQ ID NO: 32 - VSQELGQRSEQ ID NO: 33 - IPENFFGV SEQ ID NO: 34 - SEDLVVQISEQ ID NO: 35 - EAIPMSIPT SEQ ID NO: 36 - ELEGRG SEQ ID NO: 37 - EEEGLGSEQ ID NO: 38 - EEEGGG SEQ ID NO: 39 - ESESEG SEQ ID NO: 40 - EFEVEGSEQ ID NO: 41 - EIEEGG SEQ ID NO: 42 - ERESTG SEQ ID NO: 43 - EREAQGSEQ ID NO: 44 - EKETGG SEQ ID NO: 45 - EREAQG SEQ ID NO: 46 - ETEGRGSEQ ID NO: 47 - ENEAGG SEQ ID NO: 48 - EPESSG SEQ ID NO: 49 - EPESSGSEQ ID NO: 50 - ESESEG SEQ ID NO: 51 - EGEQEG SEQ ID NO: 52 - EPEPEGSEQ ID NO: 53 - EREAQG SEQ ID NO: 54 - EAEGTG SEQ ID NO: 55 - EFPEVEGSEQ ID NO: 56 - GEEGVEEG SEQ ID NO: 57 - GARGLEGSEQ ID NO: 58 - GPPGLAPG SEQ ID NO: 59 - GYPGSSRGSEQ ID NO: 60 - GFAGLPNG SEQ ID NO: 61 - GGGGSLLGSEQ ID NO: 62 - GPAGAARG SEQ ID NO: 63 - GLEGGGGGSEQ ID NO: 64 - GGGGSLLG SEQ ID NO: 65 - GFFGFPIGSEQ ID NO: 66 - EPAGAARG SEQ ID NO: 67 - GDRGLPIGSEQ ID NO: 68 - GEPEGAKG SEQ ID NO: 69 - GFKEGVEGSEQ ID NO: 70 - GVEGVELG SEQ ID NO: 71 - GFKEGVEGSEQ ID NO: 72 - GERGVLG SEQ ID NO: 73 - GGGSLLG SEQ ID NO: 74 - PEEGVEEGSEQ ID NO: 75 - GFKEGVEG SEQ ID NO: 76 - GFKEGVEGSEQ ID NO: 77 - GEPEGAKG SEQ ID NO: 78 - TEGEARGSSEQ ID NO: 79 - EGEGEGEG SEQ ID NO: 80 - EFRGVT SEQ ID NO: 81 - PRYLTASEQ ID NO: 82 - (G/P/E)XX(G/E)-ΦXXG, where Φis G, V, L, S, A, F, or T and X is any amino acid.SEQ ID NO: 83 - EXE-eXG,where e is G, V, E, A, T, S, Q, P, N, or D and X is any amino acid.SEQ ID NOs 84-143: Other Exemplary Variant Bait Region Sequences.SEQ ID NO 84: LEQYEMHGPE GLRVGKEEEG LGSIPENFFG VSELEGRGSK LSEQ ID NO 85: LEQYEMHGPE GLRVGIPENF FGVSELEGRG SKEEEGLGSK LSEQ ID NO 86: LEQYEMHGPE GLRVGSELEG RGSKEEEGLG SIPENFFGVK LSEQ ID NO 87: LEQYEMHGPE GLRVGKEEEG LGSSELEGRG STAQEAGEGK LSEQ ID NO 88: LEQYEMHGPE GLRVGIPENF FGVFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 89: LEQYEMHGPE GLRVGKEEEG LGSFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 90: LEQYEMHGPE GLRVGSELEG RGSFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 91:LEQYEMHGPE GLRVGEAIPM SIPFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 92: LEQYEMHGPE GLRVGTAQEA GEGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 93: LEQYEMHGPE GLRVGVSQEL GQRFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 94: LEQYEMHGPE GLRVGTEGEA RGSFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 95: LEQYEMHGPE GLRVGTSEDL VVQFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 96: LEQYEMHGPE GLRVGEGEGE GEGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 97: LEQYEMHGPE GLRVGGEEGV EEGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 98: LEQYEMHGPE GLRVGGARGL EGFYESDVMG RGHARLVHVE EPHTKLSEQ ID NO 99: LEQYEMHGPE GLRVGGPPGL APGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 100: LEQYEMHGPE GLRVGGEPEG AKGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 101: LEQYEMHGPE GLRVGEEEGG GFYESDVMGR GHARLVHVEE PHTKLSEQ ID NO 102: LEQYEMHGPE GLRVGGYPGS SRGFYESDVM GRGHARLVHV EEPHTKLLSEQ ID NO 103: LEQYEMHGPE GLRVGGARGL EGGFAGLPNG GEEGVEEGKLSEQ ID NO 104: LEQYEMHGPE GLRVGESESE GGGGGSLLGE FEVEGGFAGL PNGKLSEQ ID NO 105: LEQYEMHGPE GLRVGGFKEG VEGEIEEGGG FKEGVEGKLSEQ ID NO 106: LEQYEMHGPE GLRVGESESE GGFAGLPNGK EEEGLGSIPE NFFGVKLSEQ ID NO 107: EQYEMHGPE GLRVGIPENF FGVTSEDLVV QEAIPMSIPK LSEQ ID NO 108: LEQYEMHGPE GLRVGEAIPM SIPTSEDLVV QIPENFFGVK LSEQ ID NO 109: LEPAGAARGE SESEGGFFGF PIGERESTGG DRGLPIGENE AGGKLSEQ ID NO 110: LETEGRGERE AQGEFPEVEG EEEGGGPEKE TGGEREAQGK LSEQ ID NO 111: LEARGLEGGG GGSLLGGYPG SSRGGFKEGV EGGPAGAARG KLSEQ ID NO 112: LEPGLAPGGE EGVEEGGPEE GVEEGGFKEG VEGEPESSGK LSEQ ID NO 113: LEEGEARGST AQEAGEGPKE EEGLGSSELE GRGSPVSQEL GQRKLSEQ ID NO 114: LEAQEAGEGK EEEGLGSPVS QELGQRSELE GRGSPTEGEA RGSKLSEQ ID NO 115: LEEEEGLGSK EEEGLGSPKE EEGLGSKEEE GLGSPKEEEG LGSKLSEQ ID NO 116: LEELEGRGSK EEEGLGSIPE NFFGVFYESD VMGRGHARLV HVEEPHTKLSEQ ID NO 117: LEENFFGVTE GEARGSPTSE DLVVQKEEEG LGSEAIPMSI PKLSEQ ID NO 118: LEIPMSIPKE EEGLGSIPEN FFGVTEGEAR GSPTSEDLVV QKLSEQ ID NO 119: LELQQYEMHG PEGLRVGEAI PMSIPIPENF FGVKEEEGLG SKLSEQ ID NO 120: LEEEGVEEGK EEEGLGSGPA GAARGSELEG RGSPTEGEAR GSKLSEQ ID NO 121: LEPESSGEAI PMSIPTSEDL VVQIPENFFG VEAEGTGGER GVLGKLSEQ ID NO 122: LEGGGSLLGE PEPEGEREAQ GGVEGVELGG FKEGVEGEQE GRGKLSEQ ID NO 123: LESQELGQRE SESEGSELEG RGSGFKEGVE GKEEEGLGSG FFGFPIGKLSEQ ID NO 124: LEQYEMHGPK EEEGLGSSEL EGRGSEAIPM SIPTIPENFF GVVEEPHTKLSEQ ID NO 125: LEQYEMHGPS ELEGRGSIPE NFFGVEAIPM SIPTSEDLVV QIVEEPHTKLSEQ ID NO 126: LEQYEMHGPE GEGEGEGIPE NFFGVSEDLV VQISELEGRG SVEEPHTKLSEQ ID NO 127: LEQYEMHGPI PENFFGVSEL EGRGSEAIPM SIPTEGEGEG EGVEEPHTKLSEQ ID NO 128: LEQYEMHGPS ELEGRGSEAI PMSIPTKEEE GLGSIPENFF GVVEEPHTKLSEQ ID NO 129: LEQYEMHGPE AIPMSIPTEG EGEGEGIPEN FFGVSEDLVV QIVEEPHTKLSEQ ID NO 130: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVEAIPMSI PTVEEPHTKLSEQ ID NO 131: LEQYEMHGPE GEGEGEGISE DLVVQIPENF FGVKEEEGLG SVEEPHTKLSEQ ID NO 132: LEQYEMHGPE GEGEGEGIPE NFFGVSELEG RGSSEDLVVQ IVEEPHTKLSEQ ID NO 133: LEQYEMHGPI PENFFGVEGE GEGESELEGR GSSEDLVVQI VEEPHTKLSEQ ID NO 134: LEQYEMHGPS ELEGRGSIPE NFFGVKEEEG LGSSEDLVVQ IVEEPHTKLSEQ ID NO 135: LEQYEMHGPI PENFFGVSEL EGRGSSEDLV VQIKEEEGLG SVEEPHTKLSEQ ID NO 136: LEQYEMHGPK EEEGLGSIPE NFFGVSELEG RGSEGEGEGE GVEEPHTKLSEQ ID NO 137: LEQYEMHGPS EDLVVQIKEE EGLGSIPENF FGVSELEGRG SVEEPHTKLSEQ ID NO 138: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVKEEEGLG SVEEPHTKLSEQ ID NO 139: LEQYEMHGPS EDLVVQIEGE GEGEGIPENF FGVEAIPMSI PTEPHTKLSEQ ID NO 140: LEQYEMHGPE GEGEGEGIPE NFFGVEAIPM SIPTSELEGR GSEPHTKLSEQ ID NO 141: LEQYEMHGPE AIPMSIPTSE LEGRGSIPEN FFGVEGEGEG EGEPHTKLSEQ ID NO 142: LEQYEMHGPS ELEGRGSIPE NFFGVEGEGE GEGKEEEGLG SVEEPHTKLSEQ ID NO 143: LEQYEMHGPI PENFFGVSED LVVQIEGEGE GEGEAIPMSI PTEPHTKL

EXAMPLES Example 1—Generation and Selection of HEK293 Clones ExpressingRecombinant A2M

Recombinant A2M wild type sequence was expressed in HEK293F cells.Hek293F cells are plated adherently and allowed to attach overnight.Cells are transfected with XTreme Gene HP (Roche) and DNA in a 6 uLreagent: 2 ug DNA ratio. Cells are grown for 48 hours at 5% CO2 and 37degrees Celsius. Forty-eight hours after transfection media samples aretaken to confirm success of the transfection via an ELISA assay thatquantifies A2M protein. Cells are split so as to be in logarithmicgrowth phase and selection antibiotic (blasticidin) is added at 10 μg/mL(selection concentration determined experimentally). Cells are selectedin antibiotic until all of the negative control cells are dead (usuallyabout 4 to 5 days). Another media sample is taken at this point toconfirm that this newly established pool is still producing protein.Upon confirmation of protein production cells are plated at a density of˜100 cells/10 cm dish with 7.5 μg/mL blasticidin (maintenanceconcentration determined experimentally). This plating density is sparseenough that cells will be spaced far enough apart to allow each cell togrow into an individual colony. These colonies are collected usingcloning cylinders (Sigma) and plated in a 24 well plate to allow furthercell growth. Once cells become confluent in the 24 well plate an ELISAis performed on a media sample again to screen for the highest producingclone. High-expressing clones were selected and used for production ofA2M. The chosen clones were expanded and adapted to suspension (FIG. 3).Suspension adaption was completed by slowly changing the media to aserum-free media while the cells are in shaker flasks. Once the cultureis in suspension, protein can be collected by simply spinning the cellsout of the media. The A2M containing supernatants were subjected topurification for A2M. The higher cell number per volume of media resultsin a higher protein concentration per milliliter of media. High puritysamples were obtained after two chromatography methods. A yield of ˜12mg/L (adherent pool) was typical (FIG. 15).

Example 2—Inhibition of ADAMTS-5- and ADAMTS-4-Induced Damage ofCartilage with A2M

Bovine Cartilage Explants (BCEs) were treated with 500 ng/ml ADAMTS-5 orADAMTS-4 for 2 days, with a 3-fold serial dilution of purified A2M(FIGS. 7A, B). Concentration of A2M tested were 100, 33.3, 11.1, 3.7,1.2, 0.4 μg/mL. The variant A2M inhibited cartilage catabolism in aconcentration dependent manner. The IC₅₀ for inhibiting 500 ng/ml ofADAMTS-5 was calculated to be ˜7 μg/ml A2M (a 1:1 molar ratio). Maximuminhibition was observed in ˜90% with 100 μg/ml A2M (a 14:1 molar ratio).The A2M was shown to block formation of Aggrecan G3 fragments (FIGS. 7A,B) and FAC formation (FIG. 9).

Example 3—Comparison of APIC Retentate and Filtrate

Fresh cartilage was treated with APIC containing ˜7 mg/ml A2M. Cartilagecatabolism was efficiently blocked by 1% v/v of the Retentate of theAPIC production process (concentration of proteins >500 kDa in size),but not by the Filtrate (contains proteins <500 kDa), even at 5% v/v(FIG. 10). The chondroprotective effects of APIC were dose dependent.The inability of Filtrate to protect cartilage from catabolism byADAMTS-5 demonstrates that APIC concentrates >99% of the protectivefactors of autologous blood.

Example 4—A2M Inhibition of Cartilage Catabolism in an OsteoarthritisModel

Fresh cartilage was treated with TNF-α or IL-1βeta to inducechondrocytes to secrete proteases, similar to the pathology ofosteoarthritis. Cartilage catabolism is detected as increased sulfatedglycosaminoglycans (sGAG) in the culture media. Treatment withpro-inflammatory cytokines induces cartilage catabolism which treatmentwith variant A2M polypeptides block in a dose-dependent manner.

Example 5—Cytokine Profile of Monocytes Treated with Variant A2M

THP-1 monocyte cells were treated with or without variant A2M for 2 daysand the activation of the cells was monitored by secretion of cytokinesand growth factors into the medium. THP-1 did not show a change in thecytokines profile tested (FIG. 11). Similar results were seen in E6-1T-cells and SW982 fibroblast cells.

Example 6—Design and Synthesis of Tagged Wild-Type A2M ExpressionConstruct

A DNA sequence coding for the wild-type A2M precursor protein (SEQ IDNO. 1) was synthesized by GenScript based on the RefSeq amino acidsequence of human A2M precursor protein (RefSeq #NP_000005.2) (SEQ IDNO. 3). The codons used in the construct were optimized by GenScript formammalian codon usage bias, GC content, CpG dinucleotide content, mRNAsecondary structure, cryptic splicing sites, premature polyadenylationsites, internal chi and ribosome binding sites, negative CpG islands,RNA instability motifs, repeat sequences, and restriction endonucleasesites. A sequence encoding a fusion tag (DYKDDDDKGASHHHHHH (SEQ ID NO:150)) was added to the natural end of the protein sequence, followed bya STOP codon. The expression construct was given a Kpn1 restriction siteat the 5′ end and a BamH1 restriction site at the 3′ end. This constructwas cloned into a pUC57 vector. The insert encoding the expressionconstruct was extracted from the pUC57 vector via double digestion withKpn1 and BamH1 followed by agarose gel electrophoresis and gelextraction of the fragment. This insert was ligated into a pJ608mammalian expression vector (DNA 2.0) behind a cytomegalovirus (CMV)promoter (FIG. 23) and transformed into E. coli strain GC10 (GenesseeScientific). This step is performed to maintain and propagate thevector. The sequence of the expression construct was verified by DNAsequencing (Genewiz).

Example 7—Design of Acceptor Construct for Variable Bait Regions

The wild-type expression construct was mutated to allow switching ofbait region sequences by first introducing Xho1 and HindIII restrictionsites flanking the sequence encoding the bait region. This was done viatwo sequential site-directed mutagenesis reactions using the wild-typeexpression construct as the template. The sequence of the mutant“acceptor” construct was verified by DNA sequencing of the bait regionby Genewiz (SEQ ID NO 2). The corresponding amino acid sequence is SEQID No 4. The mutations in the DNA sequence necessarily result in threeamino acid substitutions in the protein Q693E on the N-terminal side ofthe bait region and T730K and V731L on the C-terminal of the baitregion. These mutations could not be avoided because the natural DNAsequence does not have restriction endonuclease sites that could be usedto remove the bait sequence. These mutations are included in the newbait regions design. The preservation of function of the acceptor mutantwas verified by its ability to inhibit trypsin (see below), and it wastested versus other proteases as part of the evaluation of the designedbait regions.

Example 8—Design and Creation of Variable Bait Region ExpressionConstructs

Novel variant bait region sequences (SEQ ID NOs: 6-30) and variant baitregions comprising one or more protease recognition sequences (SEQ IDNOs 31-83) were designed based on the known cleavage sites of humanaggrecan by ADAMTS-4, ADAMTS-5, various MMPs, and other proteases(Fosang et al., Eur. Cells and Mat., Vol. 15, 2008, pp. 11-26) (Table1). Some constructs retained part or the entirety of the wild-type A2Mbait sequence, but with an insertion of non-native amino acid sequencesincluding the variant bait regions of SEQ ID NOs: 6-30 and variant baitregions comprising one or more protease recognition sequences of SEQ IDNOs 31-83. Several pUC57 plasmids, each containing DNA insert sequencesencoding between one and six bait region sequences, were synthesized byGenScript and delivered to us as a lyophilized powder. Each insertsequence contains an Xho1 site at the 5′ end and a HindIII site at the3′ end for ligation into the acceptor construct. Each insert plasmid,along with the acceptor plasmid, was reconstituted in water and doubledigested overnight with 20 U of Xho1 and HindIII to liberate the insertsequences, and the digested plasmids were separated by electrophoresison a 1% agarose gel and visualized under UV light. Bands correspondingto the insert and acceptor length were extracted from the gel via aQiagen Qiaquick Gel Extraction Kit as per the kit instructions. Theconcentration of DNA obtained from each extraction was determined usinga Qubit fluorimeter (Invitrogen). Ligation of inserts into the region ofthe acceptor encoding the bait region was undertaken in a semi-randomfashion, by mixing the extracted insert fragment(s) from each insertvector digestion with 50 ng of digested acceptor plasmid in a 3:1 molarratio of insert:plasmid. Ligation was achieved using a Quick Ligationkit (New England Biolabs) according to the kit instructions. The mixtureof ligated plasmids was then transformed into E. coli strain GC10(Genessee Scientific) and spread onto Luria broth/agar plates containing100 mg/mL ampicillin to generate single colonies of transformants. 5 mLLuria broth cultures of individual colonies from each ligation reactionwere grown and the plasmid DNA contained within each extracted via aQiagen QiaPrep miniprep kit according to the kit instructions. Theseplasmids were sent to Genewiz for sequence confirmation using a primerthat anneals to the sequence of the A2M construct just upstream of thebait region. The individual chromatogram traces were analyzed for thepresence of heterogeneity in the sequence, and the sequences of theindividual inserts confirmed.

Example 9—Expression of A2M Variants

A2M variants were expressed in HEK293F cells (Gibco) by transienttransfection of each construct in suspension cells. Cells were grown toa density of 550,000 cells/mL in a Erlenmeyer cell culture flaskcontaining 20 mL of FreeStyle F17 medium (Invitrogen) containing 1×GlutaMax (Gibco) on a rotator at a speed of 125 rpm inside a 37° C.incubator containing an 8% CO2/air mixture. Cells were transfected bymixing 20 mg of plasmid DNA of each construct (wild-type or variant) ina 1:2 (w/v) ratio with TransIT Pro plus 10 μL TransIT Boost (Mirus) 15minutes before addition to media. Cells were maintained in the sameconditions for three days after transfection before the media containingsecreted recombinant protein was removed for protein purification (FIG.3).

Example 10—Purification of A2M Variants

Since the A2M expression construct encodes the precursor A2M protein,the expressed and processed recombinant protein is secreted into thecell culture medium via the natural A2M secretion signal. Secretedrecombinant wild-type A2M and A2M bait region variants were purifiedfrom the transfected cell culture media by Immobilized Metal AffinityChromatography using the 6×His tag (SEQ ID NO: 151) at the C-terminus ofeach construct. The media removed from the transfected cells wascentrifuged at 17,500 G for 15 minutes to remove all cells. Imidazolewas added to the clarified media to a final concentration of 10 mM. 1 mLof HisPur Cobalt resin slurry (Pierce) was added to the sample andallowed to equilibrate with shaking on a rocker at 4° C. for one hour.The beads were collected by centrifugation at 700 G for 2 minutes andthe supernatant discarded. The beads were washed three times in 10 mL ofa buffer of 50 mM Tris-Cl, 150 mM NaCl, 10 mM imidazole, pH 7.4, eachtime the beads were collected by centrifugation at 700G, and thesupernatant removed and discarded. The protein was eluted by mixing of 2mL of elution buffer (wash buffer containing 200 mM imidazole) with thebeads and centrifuging for 2 minutes at 700 G. The supernatant wascollected and retained, and the elution repeated a total of three times.The purified proteins contained in the sample were then concentrated to100 μL volume (typically between 100 μg/mL and 600 μg/mL−) using anAmicon spin filter with a NMCO of 100 KDa. During concentration theimidazole containing buffer was exhaustively exchanged for 50 mM HEPES,150 mM NaCl, 10 mM CaCl₂, 100 μm ZnCl₂, 0.05% (w/v) Brij-35, pH 7.4(HNZCB buffer). The concentration of the protein was determined usingBCA (Pierce) and 660 nm (Pierce) assays. 1 μg of each purified proteinwas mixed with reducing SDS-PAGE loading buffer, heated for five minutesat 95° C., and loaded onto a 7.5% Tris-glycine SDS-PAGE stain-free gel(Bio-rad). The gel was developed by exposing to UV light for fiveminutes, and a picture taken of the total protein bands. The purity ofthe recombinant A2M was estimated to be consistently greater than 90%across all variants and wild-type proteins (FIG. 15).

Example 11—Screening Increased Protease Inhibition by A2M Variants

Wild-type A2M protein and A2M variant polypeptides, including A2Mvariants containing the bait regions of SEQ ID NOs: 6-30 containing oneor more protease recognition sites of SEQ ID NOs 31-83, were screenedfor their comparative ability to inhibit proteolysis of a recombinantIDG fragment of human aggrecan which consist of the G1, G2, andinterglobular domains (R&D) by ADAMTS-4, ADAMTS-5, and MMP13. Screeningthe effectiveness of variants for the inhibition of each of theseenzymes was done in the same manner taking in consideration the rate ofthe proteolytic activity of each protease, such as those in Tables 3, 4aand 4b. The amount of IGD fragment in each sample was held constant at0.1 μg, whereas the amount of protease varied depending on the activityof the protease toward IGD fragment. Since each of the variants andwild-type A2M vary greatly in the kinetics of bind to each protease,some showed complete inhibition with no pre-incubation of A2M with theprotease, where others showed some inhibition if incubated with theprotease for 10 minutes, and others showed no inhibition even after apre-incubation of A2M with the protease. Two independent assays wereperformed on each A2M variant: one in which the protease, IGD fragment,and A2M were all added at the same time (no pre-incubation), and one inwhich the protease and A2M were pre-incubated at room temperature forten minutes before addition of the IGD fragment, in order to detectslower inhibitors binding to the proteases. For the experiment with nopre-incubation of protease with A2M, 5 μL of 150 nM tagged wild-type A2Mor an A2M variant in HNZCB buffer was added to a microcentrifuge tube. 5μL of 40 μg/mL IGD fragment was then added to the same tube and mixed.Finally 5 μL of 150 nM (ADAMTS-4 and ADAMTS-5, a 1:1 A2M:protease molarratio) or 75 nM (MMP13—a 2:1 A2M:protease molar ratio) protease wasadded to the tube. For the experiment with a 10 minute pre-incubation, 5μL of each A2M was mixed with 5 μL of protease 10 minutes beforeaddition of 5 μL of IGD fragment. All samples were incubated at 37° C.for one hour before being stopped by addition of 2× reducing SDS-PAGEloading buffer (Bio-rad) and heating for 5 min. at 95° C. 15 μL of eachsample was loaded onto a 7.5% Tris-Glycine Stain Free Gel (Bio-Rad) andrun at 150 V for 1 hour. Total protein was visualized and imaged underUV light as per gel instructions. The proteins were then blotted onto anitrocellulose membrane via an iBlot (Invitrogen) dry blotting systemusing a transfer time of seven minutes, blocked for one hour using TBScasein blocking solution (Bio-rad), and probed using an anti-IGDfragment goat polyclonal antibody (R&D Biosystems catalog # AF1220) at aconcentration of 0.1 μg/mL in TBS-T. The blot was washed three timeswith TBS-T and probed with an HRP-conjugated anti-goat IgG polyclonalantibody (Sigma catalog #A5420) at 0.1 μg/mL in casein blockingsolution. The blots were developed using ECL Plus chemiluminescence kits(Pierce) according to the manufacturer instructions. The Western blotswere imaged in a ChemiDoc imager system (Bio-rad). Each IGD fragmentband on the Western (intact and degraded IGD fragment) was quantifiedusing ImageLab software. The amount of degradation of IGD fragment inthe presence of each A2M variant was quantified by comparing theintensities of the degraded and intact IGD fragment bands (FIGS. 16-20),and the inhibitory capacity of each variant was compared to a wild-typeA2M sample that was prepared along with each batch of variants. Fromthis initial round of screening, eight variants were selected forfurther screening against MMP1, MMP2, MMP3, MMP8, MMP9, MMP12, andCathepsin K (all enzymes are recombinant human constructs and purchasedfrom R&D) and others, such as those in Tables, 4a, and 4b. Thecomparison of the inhibitory capacity of each variant was done by takingthe ratio of the intensity of the degraded band to the intact band withthe exception of MMP9 and MMP13 which degraded IGD fragment in such amanner that cleaved fragments did not appear on the Western blot. Inthese cases the comparison was done based solely on the intensity of theremaining intact IGD fragment band. Additionally, ADAMTS-1 and MMP7 onlycleaved the IGD fragment perceptibly; therefore, accurate inhibitionmeasurements could not be quantified. In these cases all of the variantswere judged to be essentially equivalent to wild-type with respect tothese two proteases. After evaluating all inhibition data, four variantswere selected based on improved or at least equivalent inhibitioncharacteristics against all proteases tested (FIGS. 17-21) or a mixtureof proteases known to degrade cartilage (FIG. 22).

Example 12—Screening of A2M Variants Vs. Proteases

To verify that the four selected A2M variants are still capable ofinhibiting the general proteases trypsin and chymotrypsin to a similardegree as the wild-type protein, the variants were tested in afluorescent proteolysis assay (Twining, S. S., Anal. Biochem. Vol. 143,1984, pp. 30-34). In this assay, one monitors the increase influorescence emission from a FITC-labeled protein substrate that iscaused by a proteolysis-dependent release of the fluorophore. Twoexperiments were done on each variant: one in which the molar ratio ofA2M:protease is held at 1:1, and another in which the A2M is reduced to0.5:1. 40 μL of wild-type or variant A2M at a concentration of 100 nM(for the 1:1 ratio) or 50 nM (for the 0.5:1 ratio) in HNZCB buffer wasmixed with 100 μL of bovine trypsin (Sigma) at 40 nM and incubated atroom temp for 5 minutes. Into this mixture 70 μL of 40 μg/mL FTC-caseinsubstrate (Pierce) was added, mixed, and immediately pipetted into threewells of a 384 well plate (65 μL/well) The plate was placed into a CaryEclipse fluorimeter and read in kinetic mode (single wavelength) withexcitation wavelength of 485 nm and emission wavelengths of 519 nm forfifteen minutes, during which time the rate of casein degradation by theprotease remains approximately linear. The emission intensity wasaveraged for the three sample wells, plotted vs. time, and a straightline fitted to the data from each sample and control (FIG. 18, left).The slope of the fitted line was taken as a measure of the proteaseactivity remaining in solution. Comparison of the four chosen A2Mvariants to the wild-type protein shows that the variants are allcapable of inhibiting various proteases, including trypsin andchymotrypsin approximately equally, to the wild-type A2M (FIG. 18,right).

Example 13—Preparation of Blood for Autologous Therapy

120 mL of whole human blood was obtained from a subject by venipuncture.38 mL aliquots of the blood were collected into two or more hematologiccollection bottles with a suitable volume of citrate dextrose solution A(“ACD-A”) in each collection bottle. The collection bottles withblood/ACD-A were placed into a fixed angle rotor centrifuge, andcentrifuged at predetermined velocities and times under ambienttemperature conditions. Approximately 15 mL of plasma was aliquoted fromeach tube with a serological pipette, leaving approximately 1 mL, ofplasma above the level of the buffy coat so as not to disturb theprecipitated cells. This process was repeated for the collection bottlesin one or more centrifuge spin cycles to yield a volume 45 mL of totalplasma from a total blood draw of 120 mL. The plasma was pooled into aseparate sterile hematologic collection bag. The compositions describedherein can be mixed with autograft or allograft tissue, such as bone,before administration to a subject.

Example 14—In Vitro Cartilage Degradation Assay

To test the hypotheses that cartilage catabolism caused byproinflammatory cytokines and cartilage-degrading metalloproteinases(ADAMTS) can be inhibited by preparations of Leukocyte-rich PRP (LR-PRP)or Autologous Platelet Integrated Concentrate (APIC-PRP) a controlled invitro cartilage degradation assay was performed. BCE was treated withADAMTS-5, TNF-α or IL-1β in the presence or absence of LR-PRP orAPIC-PRP. Cartilage catabolism was measured following 2 or 3 days inculture by proteoglycan release via the presence of sulfatedglycosaminoglycan (sGAG) in the media. Bovine articular cartilageexplants (BCE, 200 tit mg) were isolated from 1-1.5 year-old heifers andare equilibrated 3 days in culture. BCE cultures were treated for 3 dayswith or without a 33% (v/v) Leukocyte rich platelet-rich Plasma(LR-PRP), blood, or APIC-PRP prepared from the same patient. Proteasedigestion of cartilage with 500 ng/ml ADAMTS-5 for 2 days was inhibitedwith a 2-fold serial dilution of APIC-PRP [ED₅₀=0.1% v/v]. Forcytokine-induced cartilage catabolism, BCE was incubated 3 days in SFMwith or without 80 ng/ml human TNF-α or 8 ng/ml human IL-1β. Cartilagedegradation was inhibited with the addition of 5 mg/ml A2M or 30% (v/v)APIC-PRP. To demonstrate a dose-response curve of APIC-PRP, 3-foldserial dilutions of APIC-PRP [ED50=3% v/v] were used to inhibitTNF-α/IL-1p induced cartilage degradation. Cartilage catabolism wasmeasured in culture supernatant by proteoglycan release via the presenceof sulfated glycosaminoglycan (sGAG) using a DMMB assay with chondroitinsulphate standard curve. Cartilage degradation in 200 mg BCE was inducedby addition of LR-PRP (33% v/v), demonstrating it as a source ofcartilage catabolism. Treatment with proinflammatory cytokines (80 ng/mlTNF-α or 8 ng/ml IL-1β), ADAMTS-5 (500 ng/ml) also resulted in increasedsGAG in the medium. Addition of APIC-PRP inhibited cartilage catabolisminduced by cytokines, metalloproteinases or LR-PRP in a dose dependentmanner. The addition of LR-PRP at the highest concentration used in theAPIC-PRP study reduced but did not inhibit cartilage catabolism inducedby cytokines or MMP's measured by the release of sGAG in the medium(data not shown). Osteoarthritis (OA) is characterized by progressivedegeneration of articular cartilage. The BCE model is representative ofstudying putative therapeutics in OA. This study demonstrates thatLeukocyte-rich PRP (LR-PRP) contributed to cartilage catabolism, butAPIC-PRP protected cartilage from degradation by known OA mediators.This activity can be explained by the 5-10 fold increased concentrationof A2M in APIC-PRP over its concentration in blood. This conclusion isin agreement with experiments that demonstrate the protective effect ofA2M on cartilage. This improved understanding of cartilage biology andmetabolism should lead to clinical trials of APIC-PRP in humans.

Example 15—Chondroprotective Effect in Rabbit Model

The pathology ad osteoarthritis involves the upregulation ofinflammatory mediators and preleases such as matrix metalloproteases(MMPs) A2M is a naturally occurring plasma glycoprotein that is a potentprotease inhibitor. A2M is behaved to modulate cartilage catabolism byits ability to bind, trap and clear MMPs. Though A2M functionsthroughout multiple tissues and extracellular spaces, it does notnormally reach high levels within the intrarticular joint space. Theability of the Autologous Protease Inhibitor Concentrate (APIC-CellFree), which concentrates A2M from the blood, was tested to inhibitcartilage catabolism, and thereby attenuate the development ofosteoarthritis in a ACL-T rabbit model. The rabbit model represents afunctional load-bearing in vivo anatomical model for the evaluation ofosteoarthritis, which exhibits mechanical properties, morphologicalstructures, and healing capacity similar to human tissues. Female 8-12months old New Zealand white rabbits were used in this study. Thisrabbit model represents a functional load-bearing in vivo anatomicalmodel for the evaluation of osteoarthritis which exhibits mechanicalproperties, morphological structures and healing capacity similar tohuman tissue. Multiple Injection Cohort (Group 1): 6 rabbits receivedACL-T surgery on the right knee and sham surgery on the left knee. Fourinjections of 0.3 mL Autologous Protease Inhibitor Concentrate(APIC-Cell Free) were prepared from the rabbit blood and wereadministered on day 1. 4, 14, and 28 following the ACL knee injury.Rabbits received an equivalent volume of the sterile isotonic saline inthe contra-lateral control knee. The rabbits were monitored for 6 weeks,then sacrificed for cartilage degeneration assessment. Control Group(Group 2): 6 rabbits received ACL-T surgery on the right knee withoutsham surgery on the left knee. These rabbits were the control group andaccordingly did not receive any treatment.

Variant A2M Preparation

Prior to the ACL injury, variant A2M polypeptides were prepared. Everyrabbit received the protease inhibitor concentrate. Six weeks after theACL-T operation the animal was sacrificed for macroscopic andmicroscopic knee joint cartilage evaluation to determine OA progression.

Macroscopic and Histological Analyses

For macroscopic evaluation, the distal femoral condyles and tibialplateau surfaces were analyzed and lesions were classified using avalidated 0 to 8 scale as previously described. The locations of thelesions in the joint were recorded by a specific nine-area grid of eachjoint surface, following the classification of the InternationalCartilage Repair Society (OARSI), which was adapted to the rabbit kneeby Lindhorst et al. After macroscopic examination. Isolated femoral andtibial samples were feed and decalcified for histological (microscopicevaluation). Macroscopic evaluation of the femur and tibia demonstratedfeatures consistent with cartage catabolism consistent with OA.Treatment with APIC Cell Free considerably improved cartilageappearance, similar to the sham surgery control (FIGS. 12-14).Application of APIC reduced cartilage degradation by 53+/−20% comparedto untreated controls (mean±SEM. p=0.0086) (FIGS. 13A and 13B). Theconcentration of the variant A2M was determined. There was adose-dependent correlation between higher concentrations of A2M in anddecreased OARSI total knee score on the macroscopic evaluation (FIGS.13A and 13B). There was also a dose-dependent therapeutic benefit totreatment observed in sum OARSI histopathology evaluations of Safarin-Ostaining (r²=0.73), Structure (r²=0.76), Chondrocyte density (r²=0.50),and Cluster Formation (r²=0.97) (FIG. 14). The data suggests that theAutologous Protease inhibitor Concentrate (APIC-Cell Free), whichcontains 9-10 times the A2M concentration in blood, has achondroprotective effect on an osteoarthritis rabbit model.

Example 16—Effect of A2M on BCEs

To test the hypothesis that the addition of proinflammatory cytokines orcartilage-degrading metalloproteinases (ADAMTS and MMP) stimulatecartilage degradation that will be inhibited by A2M, a controlled invitro cartilage degradation assay was performed. Bovine CartilageExplants (BCE) were treated with or without proinflammatory cytokines(TNF-α or IL-1β) or cartilage-degrading metalloproteinases (ADAMTS-5,ADAMTS-4, MMP-7, or MMP-12) in the presence or absence of purified A2M.

Bovine articular cartilage explants (BCE. 100±4 mg) were isolated from1-1.5 year-old heifers and were equilibrated 3 days in culture. Todegrade cartilage by protease digestions, BCE was incubated 2 days inSerum-free Media (SFM) with or without 500 ng/mL ADAMTS-4 or ADAMTS-5and 3-5 μg/mL of MMP-3, MMP-7, MMP-12, or MMP-13. MMP-3 was activatedwith chymotrypsin before application on BCE. For cytokine-inducedcartilage catabolism, BCE (200+/−4 mg) was incubated 3 days in SFM withor without 80 ng/ml human TNF-α and 8 ng/mL human1L-1β. Cartilagedegradation was inhibited with the addition of 100 μg/mL of purifiedhuman A2M for protease digestion or 5 mg/mL A2M for cytokine-induceddegradation.

Cartilage catabolism was measured in culture supernatant by 1)proteoglycan release via the presence of sulfated glycosaminoglycan(sGAG) and 2) the presence of cartilage proteoglycan fragments byBio-Rad Stainless SDS-PAGE and Aggrecan G3 fragments by Westernblotting.

Fibronectin and Aggrecan Complexes (FAC) were formed by combiningdegraded cartilage matrix proteoglycans from the BCE experiments withFibronectin and Synovial Fluid and incubating for 4 hours. Newly formedFAC was measured by the FACT ELI SA, with the alteration of using anα-Aggrecan G3 antibody needed to recognize bovine aggrecan.

The IC₅₀ needed to inhibit cartilage catabolism by 500 mg/mL proteaseswas 7 μg/mL A2M for ADAMTS-5 and 3 μg/mL for ADAMTS-4. Addition of 5mg/mL A2M also inhibited cartilage catabolism induced by TNF-α or IL-1β.Further, A2M blocked production of Aggrecan G3 fragments, which formcomplexes with fibronectin and are a marker for pain and degradingjoints. (FIGS. 7-10).

Example 17—In Vitro Effect of A2M on Wound Healing

To test the hypothesis that the addition of proinflammatory cytokines orcartilage-degrading metalloproteinases (ADAMTS and MMP) slow woundhealing that will be inhibited by recombinant A2M, a controlled in vitrowound healing assay is performed. Cells from animal wounds are treatedwith or without proinflammatory cytokines (TNF-α or IL-1β) orcartilage-degrading metalloproteinases (ADAMTS-5, ADAMTS-4, MMP-7, orMMP-12) in the presence or absence of recombinant A2M compositions.Wound cells are incubated 2 days in Serum-Free Media (SFM) with orwithout 500 ng/mL ADAMTS-4 or ADAMTS-5 and 3-5 μg/mL of MMP-3, MMP-12,or MMP-13. MMP-3 is activated with chymotrypsin before application onwound cells. For cytokine-induced retardation of wound healing, woundcells are incubated 3 days in SFM with or without 80 ng/ml human TNF-αand 8 ng/mL human1L-1β. Wound healing is enhanced with the addition of100 μg/mL of purified human recombinant A2M for protease digestion or 5mg/mL recombinant A2M for cytokine-induced degradation.

Example 18—Wound Fluid Collection Technique

There are several techniques that were utilized to collect wound fluid.One technique involved aspirating wound fluid from wet wounds utilizinga syringe. Another technique involved use of a filter paper to absorbthe wound fluid, followed by extraction of the absorbed wound fluid fromthe filter paper, such as by washing with a buffer. Another techniqueinvolved running a straight edge tongue blade across the wound andcollecting the fluid that gathered in front of the straight edge, suchas with a filter paper.

For example, human chronic wound fluid is extracted from primary woundfluid dressing by soaking a single dressing overnight in 5 ml phosphatebuffered saline pH 4.0-6.0 50 mM sodium acetate adjusted to relevant pHwith glacial acetic buffer acid pH 7.0-8.0 0.2MTris(hydroxymethyl)aminomethane (Tris) corrected to buffer relevant pHusing 0.2M hydrochloric acid.

Example 19—Effects of A2M Compositions on Wound Healing in Diabetic RatsSummary

Healing of chronic wounds such as diabetic ulcers is a significantclinical problem. This study examines the in vivo response to thetherapeutic recombinant A2M compositions according to the presentinvention. The preliminary animal study on a diabetic rat model withimpaired wound healing is conducted comparing the recombinant A2Mcompositions described herein with distilled water. As a result, thetime to complete closure of wounds is lower in the A2M treated group.The difference in wound healing since day 9^(th) of the treatment isapparent. The A2M treated animals have lower scar tissues and the furgrowth is complete. In water-treated animals a scar with impaired furgrowth is apparent. The results of this study suggest that dermal use ofthese A2114 compositions have a potential to modulate wound healing andstimulate fur growth.

Methods

The animal model for in vivo testing of the recombinant A2M compositionsis a full-thickness wound in the dorsal skin of diabetic rats. Wistarrats weighing 200-250 g are used. Animals are caged in separate cages.Diabetes is induced by administration of streptozotocin (Sigma-Aldrich,UK). Streptozotocin is administered at dose of 55 mg/kgintraperitoneally. Before the administration of streptozotocin, abaseline blood glucose of rats is determined. After 48 hours, the bloodglucose is again measured to ensure rats are diabetic. The induction ofdiabetes is confirmed if the blood glucose level is doubled. Glucose isdetermined by a Glucometer (Infopia Co., Korea). Determination of bloodglucose continues every 5 days to ensure the subsistence of diabetes.Regarding the entity of streptozotocin-induced diabetes, the animalswhich lose much weight and become week, and those with uncertain bloodglucose levels are excluded from the study. A total of 14 rats are usedwith equal numbers in control and test groups. The test group has avolume of a solution comprising the recombinant A2114 compositionapplied and the control group is dressed with distilled water. At time=0days, a full-thickness, circular 15 mm diameter wound is created (e.g.,according to Wound Rep. Reg. 2002; 10: 286-294). Rats are anaesthetizedby intraperitoneal pentobarbital (55 mg/kg) and the dorsal skin isprepared for surgery using Betadine. The wound is created using surgicalscissors. At time=0 days dressings are placed, as prepared, directly onthe wounds. The wounds are covered by sterile gases and wrappedcarefully. Every 2-3 days following surgery, wounds were redressed withfresh control or test dressings while the rats were under anesthesia.The wounds are flushed with sterile saline to remove debris and to cleanthe wound area. A digital camera is used to take the pictures of thewound. The pictures are examined for wound healing in terms of woundsize and appearance of new fresh epithelium. Once photographed, freshdressings are placed on the wounds, and the wounds are covered again.Control of bias is achieved by assigning a code to each of theexperimental groups. Investigators are blinded to the identity of eachof the groups and the test and control have a similar appearance. Thecode is broken following completion of the final 4-week analysis.

In the test group on the 15^(th) day of therapy the wound is completelyclosed and the new, short fur covers the scar area. On the 22^(nd) dayof therapy the wound is completely healed and the new, long fur coversthe entire scar area. No signs of the previous wound can be seen. In thecontrol group on the 15^(th) day of therapy the wound is not closed. Onthe 22^(nd) day of testing the wound is closed but the scar is stillsever and completely naked.

Wound areas and perimeters are similar in test and control groups;however, there is a tendency for more rapid closure in the test group,particularly at day 15 where the difference in wound areas andperimeters is most pronounced. The time to complete closure of wounds islower in A2114 treated animals. In both control and test groups, woundarea begins to decrease at day 9^(th) and approximately complete woundclosure first occurs by day 15^(th) (one out of seven rats). By day22^(th), wounds are essentially closed in both groups but growth of furin the A2114 treated group is especially complete as compared to thewater-treated group.

The results of this study suggest that dermal preparation comprising therecombinant A2N compositions according to the present invention haspotential to enhance wound healing. In addition to accelerating woundclosure, A2M treatment in this study appears to improve the quality ofthe tissue in the healing wound since the fur grew more efficiently thanin the control group. Chronic wounds are not only characterized byuntimely healing and the inability to remain closed following healing.Thus, time to closure may not be the only relevant end point or solebasis for efficacy of the treatment. Obtaining the healthier scar tissuein the test group animals treated with the recombinant A2M compositionsallows anticipating a lowered recurrence rate.

Example 20—Wound Debridement

Recombinant A2M compositions are applied to necrotic tissues on pigs foran in vivo debridement efficacy study. Recombinant A2M compositions,together with a debrider, are used to each of the wounds generated(about 2 cm in diameter). After 24 hours, significant wound debridementis observed on the wounds treated with the A2M compositions. After 5days, those with recombinant A2M compositions show clean surfaceswithout any necrotic tissue and complete healing. Debrider treatedwounds also show significant debridement after 48 hours. However, thewounds are not as clean as those treated with recombinant A2Mcompositions, and did not show complete healing after five days.

What is claimed is:
 1. A composition comprising a recombinantalpha-2-macroglobulin (A2M) polypeptide comprising a non-natural baitregion, wherein the non-natural bait region comprises a sequence with atleast 80% identity to SEQ ID NO:
 20. 2. The composition of claim 1,wherein the non-natural bait region comprises a protease recognitionsequence selected from the group consisting of a matrixmetalloproteinase (MMP) or A Disintegrin and Metalloproteinase withThrombospondin Motifs (ADAMTS) recognition sequence.
 3. The compositionof claim 1, wherein the non-natural bait region comprises a proteaserecognition sequence selected from the group consisting of SEQ ID NOs:82 and
 83. 4. The composition of claim 1, wherein the recombinant A2Mpolypeptide is characterized by an enhanced inhibition of a proteasecompared to inhibition of the protease by a wild-type A2M protein,wherein the protease is selected from the group consisting of a serineprotease, a threonine protease, a cysteine protease, an aspartateprotease, a metalloprotease, a glutamic acid protease, and combinationsthereof.
 5. The composition of claim 4, wherein the enhanced inhibitionis nonspecific to the protease.
 6. The composition of claim 1, whereinthe non-natural bait region comprises a sequence with at least 85%identity to SEQ ID NO:
 20. 7. The composition of claim 1, wherein therecombinant A2M polypeptide further comprises an abnormal glycosylationsite.
 8. The composition of claim 1, wherein the recombinant A2Mpolypeptide has a longer half-life than a half-life of a wild type A2Mprotein when disposed within a subject.
 9. The composition of claim 1,wherein the non-natural bait region comprises a consensus sequence for aprotease selected from the group consisting of a serine protease, athreonine protease, a cysteine protease, an aspartate protease, ametalloprotease, a glutamic acid protease, and combinations thereof. 10.The composition of claim 1, wherein the non-natural bait regioncomprises a consensus sequence for a protease selected from the groupconsisting of a matrix metalloproteinase (MMP); A Disintegrin andMetalloproteinase with Thrombospondin Motifs (ADAMTS); chymotrypsin;trypsin; elastase; compliment factors; clotting factors; thrombin;plasmin; subtilisin; Neprilysin; Procollagen peptidase; Thermolysin;Pregnancy-associated plasma protein A; Bone morphogenetic protein 1;Lysostaphin; Insulin degrading enzyme; ZMPSTE2; ZMPSTE4; ZMPSTE24;acetylcholinesterase; and combinations thereof.
 11. The composition ofclaim 1, wherein the recombinant A2M polypeptide comprises a proteaserecognition sequence from a non-A2M protein.
 12. The composition ofclaim 1, wherein the non-natural bait region comprises a suicideinhibitor, wherein the suicide inhibitor is operable to covalentlyattach a protease to the recombinant A2M polypeptide.
 13. Thecomposition of claim 1, wherein the recombinant A2M polypeptidecomprises a sequence with at least 80% identity to SEQ ID NO:
 4. 14. Thecomposition of claim 4, wherein the wild-type A2M polypeptide comprisesthe sequence of SEQ ID NO:
 3. 15. The composition of claim 2, whereinthe MMP or ADAMTS recognition sequence is a protease recognitionsequence selected from the group consisting of SEQ ID NOs: 31-81. 16.The composition of claim 1, wherein the non-natural bait regioncomprises a sequence with at least 90% identity to SEQ ID NO:
 20. 17.The composition of claim 1, wherein the non-natural bait regioncomprises a sequence with at least 95% identity to SEQ ID NO:
 20. 18.The composition of claim 1, wherein the non-natural bait regioncomprises a sequence with at least 97% identity to SEQ ID NO:
 20. 19.The composition of claim 1, wherein the non-natural bait regioncomprises a sequence with at least 100% identity to SEQ ID NO:
 20. 20. Acomposition comprising a recombinant A2M polynucleotide, wherein therecombinant A2M polynucleotide encodes for the recombinant A2Mpolypeptide of claim
 1. 21. The composition of claim 20, wherein thenon-natural bait region encodes for a protease recognition amino acidsequence selected from the group consisting of a matrixmetalloproteinase (MMP) or A Disintegrin and Metalloproteinase withThrombospondin Motifs (ADAMTS) recognition sequence.
 22. The compositionof claim 21, wherein the MMP or ADAMTS recognition sequence is aprotease recognition sequence selected from the group consisting of SEQID NOs: 31-81.